OFFICE OF ENVIRONMENTAL HEALTH HAZARD ASSESSMENT

Proposition 65
Evidence on the Male
Reproductive Toxicity of
Perfluorononanoic Acid
(PFNA) and Its Salts andPerfluorodecanoic Acid
(PFDA) and Its Salts
October 2021

Reproductive and Cancer Hazard Assessment Branch
Office of Environmental Health Hazard Assessment
California Environmental Protection Agency

CONTRIBUTORSThe Office of Environmental Health Hazard Assessment’s (OEHHA) Reproductive andCancer Hazard Assessment Branch was responsible for the preparation of thisdocument.
Authors (listed alphabetically by last name)
Marlissa Campbell, Ph.D.
Staff Toxicologist

Ling-Hong Li, Ph.D.
Staff Toxicologist

Poorni Iyer, DVM, Ph.D., DABT
Staff Toxicologist

Francisco Moran, Ph.D.
Staff Toxicologist

Farla Kaufman, Ph.D.
Staff Toxicologist

Yassaman Niknam, Ph.D.
Staff Toxicologist

Allegra Kim, Ph.D.
Research Scientist III
Acknowledgments
The valuable support of the following OEHHA staff is also acknowledged: Nancy Firchow, MLS
for conducting the literature search and Elizabeth Boxer for assistance with the epidemiologic
data.

Internal OEHHA Reviewers
Martha S. Sandy, Ph.D., M.P.H.
Chief, Reproductive and Cancer Hazard Assessment Branch
Vince Cogliano, Ph.D.
Deputy Director, Division of Scientific Programs
David Edwards, Ph.D.
Chief Deputy Director
Director
Lauren Zeise, Ph.D.

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PREFACE
Proposition 65 1 requires the publication of a list of chemicals “known to the state” tocause cancer or reproductive toxicity. The Office of Environmental Health Hazard
Assessment (OEHHA) of the California Environmental Protection Agency maintains thislist in its role as lead agency for implementing Proposition 65. The Developmental andReproductive Toxicant Identification Committee (DARTIC) advises and assists OEHHA,
and adds chemicals to the Proposition 65 list of chemicals that cause reproductive
toxicity, as required by Health and Safety Code section 25249.8. The DARTIC serves
as the state’s qualified experts for determining whether a chemical has been clearlyshown to cause reproductive toxicity.
The Committee also provides advice and consultation regarding which chemicals
should receive their review. At their meeting in December 2020, the DARTIC
recommended that perfluorononanoic acid (PFNA) and its salts and perfluorodecanoic
acid (PFDA) and its salts be placed in a ‘high’ priority group for future listing
consideration. OEHHA selected PFNA and its salts and PFDA and its salts forconsideration for listing by the DARTIC, and in March 2021 OEHHA solicited from thepublic information relevant to the assessment of the evidence on the reproductive
toxicity of these chemicals. No information was received on either PFNA and its salts or
PFDA and its salts in response to this request. This document presents evidence
relevant to the evaluation of the male reproductive toxicity of these chemicals.
Documents presenting evidence relevant to the evaluation of these chemicals for otherreproductive toxicity endpoints (e.g., female reproductive, developmental) may be
developed in the future.

.

1 The Safe Drinking Water and Toxic Enforcement Act of 1986 (California Health and Safety Code section

5249.5 et seq.)

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TABLE OF CONTENTSPREFACE ........................................................................................................................ii
list of abbreviations..........................................................................................................ix
EXECUTIVE SUMMARY............................................................................................... xiii
Systematic Literature Review Approach .................................................................... xiii
Pharmacokinetics of PFNA and PFDA ...................................................................... xiii
Endocrine System Involvement in Development and Function of the Male
Reproductive System................................................................................................. xiv
PFNA and its salts: Male reproductive toxicity .......................................................... xiv
Studies in Humans ............................................................................................. xiv
Studies in Animals ...............................................................................................xv
Mechanistic Considerations ................................................................................ xvi
Key characteristics of male reproductive toxicants and endocrine disrupting
chemicals .......................................................................................................... xvii
PFDA and its salts: Male reproductive toxicity ........................................................ xviii
Studies in Humans ........................................................................................... xviii
Studies in Animals ............................................................................................ xviii
Mechanistic Considerations ................................................................................ xix
Key characteristics of male reproductive toxicants and endocrine disrupting
chemicals ............................................................................................................xx
1.

Introduction ........................................................................................................... 1
1.1 Identity of perfluorononanoic acid (PFNA) and its salts and perfluorodecanoic acid
(PFDA) and its salts ..................................................................................................... 1
1.2 Uses, occurrence, and exposure ........................................................................... 2
1.3 Reviews by other health agencies ......................................................................... 7
1.4 Overview of systematic literature review approach ................................................ 8
Search process..................................................................................................... 8
Data sources: ....................................................................................................... 8
Literature screening process ................................................................................ 92.

Pharmacokinetics of PFNA and PFDA ............................................................... 10
2.1 Absorption ........................................................................................................... 10

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2.2 Distribution ........................................................................................................... 10
Transport ............................................................................................................ 11
Maternal-fetal transfer ........................................................................................ 11
Breast milk transfer............................................................................................. 12
Sex differences in distribution ............................................................................. 12
2.3 Metabolism .......................................................................................................... 13
2.4 Excretion .............................................................................................................. 13
2.5 Species differences in PFNA and PFDA serum half-lives .................................... 13
PFNA .................................................................................................................. 13
PFDA .................................................................................................................. 14
3.

Endocrine System Involvement in Development and Function of the Male
Reproductive System .................................................................................... 14
3.1 Hypothalamus-pituitary-gonad axis...................................................................... 14
3.2 Thyroid hormones ................................................................................................ 15

4.

PFNA and its salts: Male reproductive toxicity .................................................... 16
4.1 PFNA: Human studies of male reproductive effects ............................................ 16
Anogenital distance ............................................................................................ 16
Male reproductive function ................................................................................. 17
Cancer ................................................................................................................ 18
4.2 PFNA: Animal studies of male reproductive toxicity............................................. 29
Organ Weight and Histopathology ...................................................................... 30
Sperm Parameters ............................................................................................. 34
Hormonal Effects ................................................................................................ 34
Fertility or Reproductive Performance ................................................................ 36
Development of the Male Reproductive System ................................................. 37
4.3 PFNA: Mechanistic considerations and other relevant data ................................ 46
General toxicity ................................................................................................... 46
In vitro study in testicular cells ............................................................................ 46
Effects on the hypothalamic-pituitary-gonadal-(liver) axis .................................. 46
Effects on the thyroid .......................................................................................... 56
Possible involvement of peroxisome proliferator-activated receptors ................. 63

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4.4 PFNA: Summary of evidence on male reproductive toxicity ................................ 63
Human studies.................................................................................................... 63
Animal studies .................................................................................................... 64
Coherence of results in human and animal studies ............................................ 65
Mechanistic considerations................................................................................. 66
Key characteristics of male reproductive toxicants and endocrine-disrupting
chemicals ........................................................................................................... 68
5.

PFDA and its salts: Male Reproductive Toxicity ................................................. 74
5.1 PFDA: Human studies of male reproductive effects ............................................ 74
Anogenital distance ............................................................................................ 74
Male reproductive function ................................................................................. 75
Cancer ................................................................................................................ 75
5.2 PFDA: Animal studies of male reproductive toxicity............................................. 82
Organ weights & histopathology ......................................................................... 82
Sperm parameters .............................................................................................. 86
Hormonal effects ................................................................................................ 86
Effects on fertility ................................................................................................ 87
5.3 PFDA: Mechanistic considerations and other relevant data ................................ 93
General toxicity ................................................................................................... 93
Cytotoxicity ......................................................................................................... 93
Effects on the hypothalamic-pituitary-gonadal-(liver) axis .................................. 94
Effects on the thyroid ........................................................................................ 102
Possible involvement of peroxisome proliferator-activated receptors ............... 110
5.4 PFDA: Summary of evidence on male reproductive toxicity ............................. 110
Human studies.................................................................................................. 110
Animal studies .................................................................................................. 111
Coherence of results in human and animal studies .......................................... 111
Mechanistic considerations............................................................................... 112
Key characteristics of male reproductive toxicants and endocrine-disrupting
chemicals ......................................................................................................... 113

6.

REFERENCES ................................................................................................. 117

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Appendix A. Literature Search Approach on the Male Reproductive Toxicity of PFNA
and Its Salts and PFDA and Its Salts .......................................................... 128
Search process ........................................................................................................ 128
Data sources: .......................................................................................................... 128
Literature screening process ................................................................................... 129
Use of Health Assessment Workspace Collaborative .............................................. 129

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LIST OF TABLES
Table 1.1 Identifiers for PFNA and some salts. ............................................................... 1
Table 1.2 Identifiers for PFDA and some salts. ............................................................... 2
Table 1.3 PFNA serum concentrations (ng/ml) in studies of California residents. ........... 4
Table 1.4 PFDA serum concentrations (ng/ml) in studies of California residents. ........... 6
Table 4.1 PFNA: Epidemiologic studies of male reproductive toxicity. .......................... 19
Table 4.2.1 PFNA or Wyeth-14,643: Relevant key findings in male rats treated bygavage for 28 days (NTP 2019) ............................................................................. 31
Table 4.2.2 PFNA: Evidence on the male reproductive toxicity in animal studies. ........ 38
Table 4.3.1 PFNA:......................................................................................................... 50
Table 4.3.2 PFNA Effects on thyroid hormones. ........................................................... 59
Table 4.4.2 Key characteristics of endocrine-disrupting chemicals ............................... 70
Table 5.1 PFDA: Epidemiologic studies of male reproductive toxicity. .......................... 76
Table 5.2.2 PFDA: Evidence on the male reproductive toxicity in animal studies. ........ 88
Table 5.3.1 PFDA: Effects on the hypothalamic-pituitary-gonadal-(liver) axis in animals
and in vitro studies. ................................................................................................ 97
Table 5.3.2 .PFDA: Effects on thyroid hormones. ...................................................... 105
Table A. 1 Human DART Study Searches ................................................................. 131
Table A. 2 Animal DART Study Searches ................................................................... 137

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LIST OF FIGURES
Figure 1.1 Structure of PFNA ......................................................................................... 1
Figure 1.2 Structure of PFDA ......................................................................................... 1

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LIST OF ABBREVIATIONS
Abbreviation

Full name

β
µM
11-KT
17β-HSD
3β-HSD
6:2 Cl-PFESA
8:2 Cl-PFESA
ACTH
AGD
AGDAP
AGDAS
AhR
AMH
AR
ATSDR
Bax
Bcl-2
Bcrp1
BMI
BZRP
CASA
CI
CYP11a
CYP17
CYP19aDARTDARTIC
DFI
DFI
DGML
DHT
Dpf
E2
ECHA
EDCs
EGR 3
ER
ERα
ERβ
Et-PFOSA-AcOH

regression coefficient
micromolar
11-ketotestosterone
17β-Hydroxysteroid dehydrogenase
3β-Hydroxysteroid dehydrogenase
6:2 chlorinated polyfluorinated ether sulfonate
8:2 chlorinated polyfluorinated ether sulfonate
adrenocorticotropic hormone
anogenital distanceanopenile distanceanoscrotal distancearylhydrocarbon receptor
antimüllerian hormone or Müllerian inhibiting substanceandrogen receptor
Agency for Toxic Substances and Disease RegistryBcl2 associated X
B-cell lymphoma 2
breast cancer resistance protein
body mass index
benzodiazepine receptor
computer-aided sperm analysis
confidence interval
cytochrome P450 family 11 subfamily A
cytochrome P450 family 17
cytochrome P450 family 19 subfamily A
developmental and reproductive toxicity/toxicant
Developmental and Reproductive Toxicant Identification Committee
DNA fragmentation index
DNA fragmentation index
DNA global methylation
dihydrotestosterone
days post-fertilization
estradiol
European Chemicals Agency
endocrine-disrupting chemicals
early growth receptor 3
estrogen receptor
estrogen receptor alphaestrogen receptor beta
2-(N-Ethyl-perfluorooctane sulfonamido) acetic acid

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Abbreviation

Full name

FAI
FasL
FCM
FDA
FDR
FSH
FSHR
GD
GHR
GLUT-3
GnRH
GST
GWG
HAWC
hCG
HDS
hERα

free androgen index
Fas ligand
flow cytometric
Food and Drug Administration
false discovery rate p-value
follicle-stimulating hormone
follicle-stimulating hormone receptor
gestation day
growth hormone receptor
glycose transporter 3
gonadotrophin-releasing hormone
glutathione-s-transferase
gestational weight gain
Health Assessment Workspace Collaborative
human chorionic gonadotropin
high DNA stainability
human estrogen receptor α

HMG CoA synthase 1
HO-1
HPG
HPGL
HPT
HSA
i.p.
IC50
IGF-1
IGF-1R
IKKβ
IQR
IVF
KCs
LH
LHDC
LHR
Ln
LOD
LOQ
Me-PFOSA-AcOH
MIS
mRNA

3-hydroxy-3-methylglutaryl coenzyme A synthase 1
heme oxygenase-1
hypothalamus-pituitary-gonad
hypothalamic-pituitary-gonadal-liver
hypothalamic-pituitary-thyroid
human serum albumin
intraperitoneal
half maximal inhibitory concentration
insulin like growth factor 1
insulin like growth factor 1 receptor
inhibitor of NF-κB kinase
interquartile range
in vitro fertilization
key characteristics
luteinizing hormone
lactate dehydrogenase type cluteinizing hormone receptor
natural log
limit of detection
limit of quantification
2-(N-Methyl-perfluorooctane sulfonamido) acetic acidMüllerian inhibiting substancemessenger ribonucleic acid

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Abbreviation

Full name

MTT
N
NF-κB
NHANES
NIOSH
Nrf2
NTP
OD
OEHHA
OR
PCNA
PFASs
PFBS
PFDA
PFDoA
PFHpA
PFHpS
PFHxA
PFHxS
PFNA
PFOA
PFOS
PFPA
PFTrA
PFUnDA
p-mTOR
PND
PPAR
PPARα
ppm
PSA
PVDF
qRT-PCR
r
RAC
ROS
RP
RR
rT3
SCSA
SD

3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5 diphenyltetrazolium bromide
number of participants
nuclear factor kappa B
National Health and Nutrition Examination Survey
National Institute for Occupational Safety and Healthnuclear factor-erythroid-2-related factor-2
National Toxicology Program
optical density
Office of Environmental Health Hazard Assessment
odds ratio
proliferating cell nuclear antigen
per- and polyfluoroalkyl substances
perfluorobutane sulfonic acidperfluorodecanoic acidperfluorododecanoic acidperfluoroheptanoic acidperfluoroheptane sulfonic acidperfluorohexanoic acidperfluorohexane sulfonic acidperfluorononanoic acidperfluorooctanoic acidperfluorooctane sulfonic acidperfluorophosphonic acidperfluorotridecanoic acidperfluoroundecanoic acidphosphorylated mammalian target of rapamycin
postnatal day
peroxisome proliferator-activated receptor
peroxisome proliferator-activated receptor alphaparts per million
prostate-specific antigen
polyvinylidene fluoride
quantitative real-time PCR
correlation coefficient
Committee for Risk Assessment [ECHA]
reactive oxygen species
relative potency
relative risk
reverse triiodothyronine
sperm chromatin structure assay
Sprague Dawley or standard deviation

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Abbreviation

Full name

SF1
SHBG
SOD
SR-B1
StAR
T
T3
T4
TBG
TCDD
TRs
TRH
TSH
TSPO
TTR
TUNEL
US EPA
VCL
VSL
VtG
WT1

steroidogenic factor 1
sex hormone-binding globulin
superoxide dismutase
scavenger receptor class B type 1steroidogenic acute regulatory protein
testosterone
triiodothyronine or tri-iodo-L-thyronine
thyroxine
thyroid binding globulin
2,3,7,8-tetrachlorodibenzo-p-dioxin
thyroid hormone receptors
thyrotropin-releasing hormone
thyroid stimulating hormone
mitochondrial translocator protein, also peripheral-type benzodiazepine receptor
transthyretin
terminal deoxynucleotidyl transferase dUTP nick end labeling
US Environmental Protection Agency
curvilinear velocity
straight-line velocity
vitellogenin
Wilms’ tumor gene

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EXECUTIVE SUMMARY
This document presents evidence relevant to the evaluation of the male reproductive
toxicity of perfluorononanoic acid (PFNA) and its salts and perfluorodecanoic acid
(PFDA) and its salts. PFNA and PFDA are perfluoroalkyl carboxylic acids containing
nine and ten carbons, respectively, and are members of a large group of substanceswith surfactant properties collectively called per- and polyfluoroalkyl substances(PFASs). PFNA and its salts and PFDA and its salts have been used in various
industries, including as processing aids in fluoropolymer manufacture. PFNA and PFDA
are also used in some cosmetic products. PFASs, including PFNA and PFDA, areglobal environmental pollutants of air, water, soil and wildlife, and are very persistent in
the environment. Recent data from Biomonitoring California(https://biomonitoring.ca.gov) have shown that PFNA and PFDA are readily detected in
virtually all Californians. See Section 1.2 for additional information on uses, occurrence
and exposure to PFNA and its salts and PFDA and its salts.
The evidence summarized in this document includes studies of PFNA and PFDA, as
well as studies of their salts, which dissociate to form the corresponding organic acids.
Systematic Literature Review Approach
Using a systematic approach, the Office of Environmental Health Hazard Assessment(OEHHA) conducted literature searches on the developmental and reproductive toxicityof PFNA and its salts, and PFDA and its salts, and then focused on literature relevant tomale reproductive toxicity (last comprehensive search, February 2021). An overview ofthe systematic literature review approach is presented in Section 1.4 of this document,
and more detailed information can be found in Appendix A.
Pharmacokinetics of PFNA and PFDAPFNA and PFDA are not known to be metabolized in animals or humans. PFNA andPFDA are well absorbed with oral administration in animals, bind to serum proteins, andare widely distributed throughout the body in both humans and animals. The estimatedhalf-lives of PFNA and PFDA are significantly longer in humans (3.1 years and 7.1
years, respectively) than in rodents (30-55 days and 36-109 days, respectively). In a
study of PFASs in human tissues, the highest levels of PFNA were observed in brain
and kidney, with lower levels in lung and liver, and for PFDA the highest levels wereobserved in the brain, with lower levels in lung and kidney. PFNA and PFDA can crossthe blood brain barrier and the placenta, and both have been detected in fetal tissues,
cord serum, and breast milk. PFNA and PFDA excretion pathways in humans include
urinary and fecal excretion and incorporation into nails and hair. See Section 2 for moredetailed information on pharmacokinetics.
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Endocrine System Involvement in Development and Function of the Male
Reproductive System
Reproductive biology is under close control from the endocrine system. Environmental
factors, including exposures to certain chemicals, may influence the endocrine systemand the reproductive processes controlled by it. An overview of the role of the
hypothalamus-pituitary-gonad axis and the thyroid hormone system in male
reproductive system development and function is presented in Section 3 of thisdocument.
PFNA and its salts: Male reproductive toxicity
Studies in Humans
OEHHA identified 17 epidemiologic studies of effects of PFNA on the male reproductive
system. The levels of exposure to PFNA in the available epidemiologic studies weregenerally low, with little variability in concentrations.
The epidemiologic evidence for an effect of PFNA on anogenital distance (AGD) was
mixed, with one study reporting an association with longer anoscrotal distance (AGDAS),
and another reporting non-significant associations with shorter AGDAS and anopenile
distance (AGDAP) that did not remain at 12 months of age.
Three of seven studies of effects of PFNA levels on serum testosterone (T) levels
reported decreases (two of which were statistically significant), while the other fourstudies reported inconsistent results across locations or no associations with T.
A small study with relatively high plasma PFNA concentrations and variability reported a
substantial reduction in sperm concentration, while other studies reported noassociations with sperm concentration or count. Sperm quality studies reported
decreases, increases, and no change in the proportion of sperm with normal
morphology. A study that examined both semen and serum PFNA concentrations
reported that semen PFNA was associated with decreased motility, curvilinear velocity(VCL), and straight line velocity (VSL), while other studies that used serum PFNA
measurements reported no associations with these parameters.
Compromised sperm DNA integrity was associated with semen PFNA in a study whereinfertile men were overrepresented, but not in a study using serum PFNA and in which
infertile men were underrepresented.
PFNA exposure was not associated with prostate cancer or prostate-specific antigen(PSA) concentration.
See Section 4.1 for more detailed information on male reproductive toxicity studies inhumans.
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Studies in Animals
PFNA has been evaluated for its male reproductive toxicity in a number of animalstudies in vivo, including four studies in rats, five studies in mice and one study inzebrafish. All the studies in rats or mice treated the animals by oral gavage, and thestudy in zebrafish exposed the animals to PFNA for 180 days. Two of the studies inmice evaluated the effects of PFNA on development of the male reproductive systemafter gestational treatment.
PFNA induced dose-dependent reductions in epididymal weight at ≥0.615 mg/kg-day
and testis weight at ≥1.25 mg/kg-day in adult Sprague-Dawley (SD) rats exposed for 28
days. Treatment with PFNA at ≥2 mg/kg-day for 14 days resulted in an apparent
decrease in testis weight in prepubertal mice, but the reduction did not reach statisticalsignificance.
Histopathological changes in the testis (e.g., germ cell degeneration) were observed inthree rat studies and three mouse studies. Testicular lesions observed in rats treatedwith PFNA at ≥2 mg/kg-day for 14 days included cell degeneration in spermatocytes
and spermatogonia, and cytoplasmic vacuolization in Sertoli cells. In the NTP study inrats, exposure to PFNA for 28 days caused dose-related increases in germ celldegeneration (≥2.5 mg/kg-day), interstitial cell atrophy (≥2.5 mg/kg-day), and spermatid
retention (≥2.5 mg/kg-day). In prepubertal mice, treatment with PFNA at ≥2 mg/kg-day
for 14 days or 0.5 mg/kg-day for 90 days resulted in increased germ cell degeneration
as well as other changes consistent with these effects, e.g., decreased relative
population size of 4C germ cells and decreased spermatogonial cells in G2 phase.
In studies that measured sperm parameters, a dose-dependent reduction in epididymal
sperm counts was observed in rats treated with PFNA for 28 days (≥ 1.25 mg/kg-day),and in mice dose-related reductions in sperm counts, motility and viability wereobserved following exposure to PFNA at 0.2 and 0.5 mg/kg-day for 90 days.
Reduced serum levels of T were consistently observed in rats and mice at dosing levels
that caused histopathological lesions or changes in sperm parameters. On the other
hand, increased serum T levels were observed at 1 mg/kg-day dosing level (butdecreased T at 5 mg/kg-day) in pubertal rats treated for 14 days and in zebrafish
treated for 180 days at 0.01 mg/L in water (but not 0.1 and 1.0 mg/L exposure levels).
Two studies provided limited data on the male fertility effects of PFNA, one study inmice and another in zebrafish. In mice, reductions in fertility index and litter size wereobserved when PFNA-exposed males were mated with unexposed females, but
detailed data and information on the design of the fertility assessment studies were notreported. In the zebrafish study both males and females were exposed to PFNA prior tomating, and reductions in egg production and hatching rate were observed with PFNA
treatment.
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Gestational exposure to PFNA at 5 mg/kg-day reduced intratesticular levels of T in miceon postnatal day (PND) 3. In this same study gestational exposure to PFNA at 2 and 5
mg/kg-day reduced testicular protein levels of steroidogenic acute regulatory protein(StAR) and WT1, and at 5 mg/kg-day reduced testicular protein levels of SF1,
CYP11A1, 3β-HSD, 17β-HSD, and PCNA (a marker of cell proliferation). These
proteins are critical for either steroidogenesis in Leydig cells or for Sertoli cell function
and proliferation during the perinatal period. In another study of male mice, a doserelated delay in preputial separation was observed following gestational exposures to
PFNA at 3.0 and 5.0 mg/kg-day.
See Section 4.2 for more detailed information on male reproductive toxicity studies inanimals.
Mechanistic Considerations
Potential mechanistic pathways involved in PFNA mediated male reproductive toxicity
include effects on the HPG axis, and effects on thyroid homeostasis.
HPG axis
Altered hormone levels have been observed in rats, mice, zebrafish, and primary rat
Sertoli cell cultures following exposure to PFNA. PFNA reduced plasma levels of T in
male rats and plasma and intratesticular levels of T in male mice. In male rats andzebrafish PFNA increased serum levels of estradiol (E2). In addition, PFNA increased
serum Müllerian inhibiting substance (MIS) in rats, increased MIS messenger
ribonucleic acid (mRNA) in primary rat Sertoli cells, altered testicular gene expressionof MIS in mice, and increased brain mRNA levels of the gene encoding luteinizing
hormone (LH) in male zebrafish.
PFNA induced changes in gene and protein expression of a number of enzymes andfactors involved in steroidogenesis in mice and zebrafish, and decreased steroid
hormone production in a mouse Leydig tumor cell line. In mice PFNA decreased
testicular protein levels of steroidogenic factor 1 (SF1), StAR, cytochrome CYP11a,3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase(17β –HSD), and testicular mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A
synthase 1 (HMG Co synthase 1), StAR, cytochrome P450 family 11 subfamily a(CYP11a) and 3β-HSD. In zebrafish PFNA decreased gonadal gene expression of 3βhsd, and increased gene expression of star, cyp11a, 17β-hsd, and cyp19a. In a mouse
Leydig tumor cell line, PFNA decreased steroid hormone production at concentrations
that also reduced mitochondrial membrane potential.
Receptor binding studies and in silico modeling indicate that PFNA can interact withestrogen receptors, and estrogen receptor mediated effects of PFNA have been
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observed in zebrafish, trout, and human cell lines. In fish PFNA increased vitellogenin(vtg) gene and protein expression. In human embryonic kidney cells transfected with ahuman estrogen receptor alpha (hERα) reporter gene PFNA induced an estrogenic
response, while anti-estrogenic activity was observed in studies with human breast
adenocarcinoma cell lines.
PFNA may also interact with androgen receptors, based on observations of anti-AR
activity in a Chinese hamster ovary (CHO) cell line.
Studies in rats, mice, zebrafish and primary rat Sertoli cell cultures indicate that PFNA
may affect gene and protein expression of some hormone receptors, growth factor
receptors, and related proteins. In rats, PFNA decreased testicular protein levels oftransferrin. In mice, PFNA reduced testicular mRNA levels for the androgen receptor(AR), growth hormone receptor (GHR), insulin-like growth factor hormone receptor 1(IGF-1R), and insulin-like growth factor 1 (IGF-1). In male zebrafish, PFNA decreased
brain mRNA levels for era, erb and ar, decreased gonadal mRNA levels for fshr and lhr,and increased liver mRNA levels for era and erb. In primary rat Sertoli cell cultures
PFNA reduced mRNA levels of follicle stimulating hormone receptor (FSHR), increasedmRNA levels of androgen binding protein (ABP), and reduced mRNA levels oftransferrin.
Thyroid homeostasis
Outcomes of both thyroid and male reproductive toxicity were assessed in rats following
28 days of treatment with PFNA. Thyroid effects were observed at the same or lower
doses than evidence of male reproductive toxicity. PFNA has been shown to interferewith thyroid hormone binding, serum levels, and function as assessed using in vivo, in
vitro, and in silico test systems. Existing data suggest a possible, but unproven,mechanistic connection between thyroid toxicity and the male reproductive toxicity of
PFNA.
See Section 4.3 for more detailed summaries of data relevant to considerations ofmechanisms of male reproductive toxicity.
Key characteristics of male reproductive toxicants and endocrine
disrupting chemicals
The available mechanistic evidence suggests that PFNA exhibits five of the key
characteristics (KCs) for male reproductive toxicants (e.g., alters germ cells; alters
somatic cells; alters production and levels of reproductive hormones; alters hormone
receptors; is genotoxic), and seven of the KCs for endocrine-disrupting chemicals (e.g.,interacts with or activates hormone receptors; antagonizes hormone receptors; alters
hormone receptor expression; alters signal transduction in hormone responsive cells,
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factors and transcripts and activity; alters hormone synthesis; alters hormone
distribution or circulating levels; alters fate of hormone producing or hormone
responsive cells). See Section 4.4 for more information on the data relevant to these
KCs.
PFDA and its salts: Male reproductive toxicity
Studies in humans
Eleven epidemiologic studies of effects of PFDA on the male reproductive system wereidentified. Most of the PFDA exposure concentrations in the available epidemiologic
studies were very low, with little variability.
The epidemiologic evidence for an association between maternal PFDA exposure andanogenital distance (AGD) was mixed, with the stronger of two studies reporting
associations with shorter anoscrotal distance (AGDAS) and anopenile distance (AGDAP)
at birth, but not at 12 months. The other study reported that prenatal PFDA exposure in
the third quartile, compared to the first quartile, was associated with an increase in
AGDAS at three months.
Higher serum PFDA was associated with lower serum T in a small sample of adolescentboys, while studies of adult males did not report consistent associations withreproductive hormones.
Semen quality studies with the highest PFDA concentrations reported someassociations with poorer semen quality. The largest study, which had relatively highserum PFDA concentrations, overrepresented infertile men, and measured PFDA inboth serum and semen, reported that semen PFDA was associated with decreased
progressive motility and straight line velocity (VSL). In this study semen PFDA was also
associated with increases in markers of sperm chromatin immaturity and DNA
fragmentation. In a small cohort of couples with female factor infertility, plasma PFDA
was associated with decreased sperm concentration and a nonsignificant reduction in
sperm count.
One study reported an association between PFDA and prostate cancer among men witha first-degree relative with prostate cancer; this result needs corroboration.
See Section 5.1 for more detailed information on male reproductive toxicity studies inhumans.
Studies in Animals
There are a number of animal studies in vivo that evaluated the male reproductive
effects of PFDA, including multiple studies in male rats, one study in male mice, one
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study in male hamsters, one study in male guinea pigs, and one study in zebrafish. One
study by the National Toxicology Program (NTP) treated male SD rats at five doselevels (0.156-2.5 mg/kg-day) by daily gavage for 28 days. All other rat studies assessed
the effects of PFDA seven to 30 days after administration of a single intraperitoneal
injection (i.p.) of relatively high doses (ranging from 20-80 mg/kg). The studies in malemice, hamsters, and guinea pigs also treated the animals with a single i.p. injection ofrelatively high doses of PFDA and evaluated the toxic effects 14 to 28 days after dosing.
The study in zebrafish exposed the animals for 120 days.
PFDA reduced testis weight in rats, hamsters and guinea pigs following a single i.p.
injection at relatively high doses ranging from 50-175 mg/kg. Dose-related reductions intestis and epididymis weights were observed in the NTP study in rats exposed to lower
doses for 28 days (≥1.25 mg/kg-day).
Histopathological changes in the testis (i.e., germ cell degeneration) were observed inrats (in two studies), hamsters, and guinea pigs that received a single i.p. dose of ≥50
mg/kg (or 150 mg/kg for guinea pigs). Testicular lesions observed in rats exposed tolower doses for 28 days included a dose-dependent increase in interstitial cell atrophy(≥1.25 mg/kg-day), and increased spermatid retention or inhibited spermiation (2.5mg/kg-day. No effects on testis weight or histology were observed in mice on day 28
following a single i.p. dose of 250 mg/kg PFDA.
There is one study that measured sperm parameters in rats. In this study, the authors
found a dose-dependent reduction in epididymal sperm counts in rats exposed to PFDA
for 28 days, which was significant at 2.5 mg/kg-day. Reduced serum levels of T wereobserved in rats exposed to PFDA for 28 days (dose-dependent; statistically significant
at ≥1.25 mg/kg-day) and in rats receiving a single dose (≥40 mg/kg). Plasma
dehydrotestosterone (DHT) levels were also decreased by PFDA in the single dose rat
study. The ratios of blood E2/T and E2/110-KT were increased in zebrafish exposedfrom one to 120 days post fertilization to PFDA at 1.0 mg/L, but not at higher or lower
concentrations. There are no animal studies available on the male fertility effects of
PFDA. See Section 5.2 for more detailed information on male reproductive toxicity
studies in animals.
Mechanistic Considerations
Potential mechanistic pathways involved in PFDA mediated male reproductive toxicity
include effects on the HPG axis, and effects on thyroid homeostasis.
HPG axis
PFDA altered steroid hormone levels in rats, decreased steroid hormone synthesis in
isolated rat Leydig cells, mouse Leydig tumor cell lines, and rat testes ex vivo and
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altered aromatase gene or protein expression in zebrafish and, a human cell line. In
male rats, PFDA reduced plasma levels of T and DHT without effects on plasma levelsof LH and caused a dose-dependent decrease in hCG-stimulated T secretion in
decapsulated testes from PFDA treated animals, indicating disruption of testicular
feedback to LH stimulation. In vitro, exposure to PFDA resulted in decreases in steroid
hormone production in isolated rat Leydig cells and mouse Leydig tumor cell lines.
PFDA reduced translocator protein (TSPO) protein levels and mRNA stability, anddecreased mitochondrial membrane potential in mouse Leydig tumor cell lines,
suggesting that impairment of mitochondrial function and cholesterol transport may
contribute to decreased steroidogenesis. PFDA increased gonadal aromatase gene
expression in male zebrafish, while it decreased aromatase activity levels in a humanplacental choriocarcinoma cell line.
Receptor binding studies and in silico modeling indicate that PFDA can interact withestrogen receptors, and estrogen receptor mediated effects of PFDA have been
observed in zebrafish, trout, and human cell lines. In fish PFDA increased vtg gene andprotein expression. In human embryonic kidney cells transfected with a hERα reporter
gene PFDA induced an estrogenic response, while anti-estrogenic activity wasobserved in studies with human breast adenocarcinoma cell lines.
PFDA may also interact with androgen receptors, based on observations of anti-AR
activity in a Chinese hamster ovary cell line.
Thyroid homeostasis
Outcomes of both thyroid and male reproductive toxicity were assessed in rats following
28 days of treatment with PFDA. Thyroid effects were observed at the same or lower
doses than evidence of male reproductive toxicity. PFDA has been shown to interferewith thyroid hormone binding, serum levels, and function as assessed using in vivo, in
vitro, and in silico test systems. Existing data suggest a possible, but unproven,mechanistic connection between thyroid toxicity and the male reproductive toxicity of
PFDA.
See Section 5.3 for more detailed summaries of data relevant to considerations ofmechanisms of male reproductive toxicity.
Key characteristics of male reproductive toxicants and endocrine
disrupting chemicals
The available mechanistic evidence suggests that PFDA exhibits four of the key KCs formale reproductive toxicants (e.g., alters germ cells; alters somatic cells; alters
production and levels of reproductive hormones; alters hormone receptors), and six of
the KCs for endocrine-disrupting chemicals (e.g., interacts with or activates hormone
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receptors; antagonizes hormone receptors; alters signal transduction in hormone
responsive cells, factors and transcripts and activity; alters hormone synthesis; alters
hormone distribution or circulating levels; alters fate of hormone producing or hormone
responsive cells). See Section 5.4 for more information on the data relevant to these
KCs.

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1. INTRODUCTION
1.1 Identity of perfluorononanoic acid and its salts and perfluorodecanoic acidand its salts
Perfluorononanoic acid (PFNA) and its salts and perfluorodecanoic acid (PFDA) and its
salts are perfluorinated organic compounds with surfactant properties, and members ofa large group of substances collectively called per- and polyfluoroalkyl substances(PFASs). PFNA and PFDA are perfluoroalkyl carboxylic acids containing nine and 10
carbons, respectively (see Figure 1.1 and Figure 1.2). Perfluoroalkyl carboxylic acids
exist in equilibrium with the ionic form of the molecule. Table 1.1 and Table 1.2
summarize key identifiers for PFNA, PFDA, and some of their salts.

Figure 1.1 Structure of PFNA

Figure 1.2 Structure of PFDA
Table 1.1 Identifiers for PFNA and some salts.
Chemical name
Perfluorononanoicacid

AbbreviationPFNA

IUPAC name

Molecular
formulaCAS
RN

Heptadecafluorononanoic
acid

C9HF17O2

375-951

Perfluorononanoate PFNA ion

Heptadecafluorononanoate C9F17O2

Ammoniumperfluorononanoate

Ammonium
heptadecafluorononanoate

Ammonium
PFNA;
PFNA-NH4+

7200768-2

C9H4F17NO2 414960-4

Abbreviations: IUPAC: International Union of Pure and Applied Chemistry; CAS RN: Chemical Abstracts
Service Registry Number

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Table 1.2 Identifiers for PFDA and some salts.
Chemical name

Abbreviation

Perfluorodecanoic
acid

PFDA

IUPAC name

Molecular
formulaCAS RN

Nonadecafluorodecanoic
acid

C10HF19O2

335-76-2

Perfluorodecanoate PFDA ion

Nonadecafluorodecanoate C10F19O2

Sodiumperfluorodecanoate

Sodiumnonadecafluorodecanoate

SodiumPFDA;
PFDA-Na+7382936-4

C10F19NaO2 3830-453

Abbreviations: IUPAC: International Union of Pure and Applied Chemistry; CAS RN: Chemical Abstracts
Service Registry Number

1.2 Uses, occurrence, and exposure
PFASs are commonly used to make products resistant to stains, grease, soil and water,
and are used in various industries, often as processing aids. For example, ammonium
PFNA has been used as a processing aid in the emulsion process used to makefluoropolymers such as polyvinylidene fluoride (PVDF) (Prevedouros et al. 2006).
Surflon S-111, which contains approximately 74% ammonium PFNA, is a commercial
PFAS mixture commonly used for this purpose (NJ Drinking Water Quality Institute2015). In 2006 the residual content of ammonium PFNA in PVDF was estimated torange from 100 to 200 parts per million (ppm) (Prevedouros et al. 2006).
PFASs are used in cosmetic products, such as creams, lotions, concealers,
foundations, body lotions, and sunscreens. PFNA and PFDA have been detected incosmetic products, including those purchased in Japan, Sweden, and Denmark (Danish
EPA 2018). In a recent study, PFDA was detected in five of 17 cosmetic products
purchased in Canada (two foundation products, two lip products, and one mascara) and
one of 12 cosmetic products purchased in the US (one foundation product) (Whitehead
et al. 2021).
Production of ammonium PFNA, used primarily as a processing aid in fluoropolymer
manufacture, is thought to have started around 1975 (Prevedouros et al. 2006). The
estimated global production in 2004 of PFNA and ammonium PFNA was estimated as
between 15 to 75 tons, and the total global historical production of PFNA andammonium PFNA from 1975 to 2004 was estimated to be between 800- 2300 tons
(Prevedouros et al. 2006). In 2006, Japan was the primary producer of ammonium
PFNA (Prevedouros et al. 2006).

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The Office of Environmental Health Hazard Assessment (OEHHA) was unable to locate
current information on production for either PFNA or PFDA. The US Environmental
Protection Agency (US EPA) has proposed a regulation that “would require all
manufacturers (including importers) of PFAS in any year since 2011 to report
information related to chemical identity, categories of use, volumes manufactured andprocessed, byproducts, environmental and health effects, worker exposure, anddisposal” (for more information follow this link: https://www.epa.gov/chemicals-undertsca/epa-continues-take-action-pfas-protect-public; the comment period for this rule wasjust extended to September 7, 2021).
PFASs, including PFNA and PFDA, are global pollutants of air, water, soil and wildlife,
and are very persistent in the environment (NJ Drinking Water Quality Institute 2015).
Estimated total global emissions of PFNA and ammonium PFNA fromperfluorocarboxylic acid manufacturing between 1975 to 2014 range from 70-200 tons,
and estimated total global emissions of ammonium PFNA from fluoropolymer
manufacture during this same period range from 400 to 1400 tons (Prevedouros et al.
2006). Other sources of PFNA emissions and exposure include the release of PFNA
(and PFOA) from the fluorotelomer alcohol, 8:2 FTOH, as a result of environmental
transformation, which can occur in the atmosphere, in soil as a result of microbial
transformation, and in fish. Fluorotelomer alcohols have been used to make greaseresistant food packaging materials and other consumer products (NJ Drinking Water
Quality Institute 2015). Results from FDA’s first survey of nationally distributed
processed foods detected PFNA in fish sticks and patties (frozen then cooked) andPFDA in tuna (canned in water) (Genualdi et al. 2021).
No emissions data were identified for PFDA.
Human biomonitoring studies indicate that exposure to PFNA and PFDA is widespread.
The tables below present data on serum concentrations of PFNA (Table 1.3) and PFDA
(Table 1.4) measured in Californians in studies conducted between 2010 and 2019
(Biomonitoring California 2020).

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Table 1.3 PFNA serum concentrations (ng/ml) in studies of California residents.

Study Name
Maternal and Infant
Environmental Exposure
Project (MIEEP)
California Teachers Study(CTS)
Firefighter Occupational
Exposures (FOX) ProjectMeasuring Analytes inMaternal Archived Samples(MAMAS)
Biomonitoring Exposures
Study (BEST) - 1. Pilot
Biomonitoring Exposures
Study (BEST) - 2. Expanded
Asian/Pacific Islander
Community Exposures(ACE) Project - ACE 1
Asian/Pacific Islander
Community Exposures(ACE) Project - ACE 2
California Regional
Exposure Study, Los
Angeles County (CARE-LA)
California Regional
Exposure Study, Region 2(CARE-2)

Geometric 95%
95%
Samplemean
Lower UpperYear(ng/ml)CI
CI
2010 to
0.733
0.621 0.865
2011

N
77

Detection
Frequency
Notreported

2011

0.92

0.89

0.95

1719

99.70%

2010 to2011
2012 to
2015

1.15

1.06

1.25

101

100%

0.647

0.596

0.703

200

100%

2011 to
2012
2013

0.994

0.92

1.07

110

100%

0.787

0.726

0.853

337

99.10%

2016

0.987

0.87

1.12

96

99%

2017

1.1

0.988

1.22

99

99%

2018

0.3

0.278

0.323

425

97.20%

2019

0.205

0.187

0.225

358

92.2%

Data available at https://biomonitoring.ca.gov/. (Biomonitoring California 2020). Abbreviations: CI:
confidence interval; N: number of participants

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Recent findings from other human biomonitoring studies of PFNA include the following:•••

The National Health and Nutrition Examination Survey (NHANES) for theyears 2009 to 2010 reported serum concentration levels (ng/ml; geometricmean and 95% confidence intervals [CI]) in males aged 20 years or older of
1.40 (1.20, 1.63) (Dobraca et al. 2015).
NHANES for the years 2013 to 2014 reported serum concentration levels(ng/ml; unadjusted geometric mean and 95% CI) in children aged 3–11 yearsof 0.79 (0.68−0.93) (Jain 2018).
A 95% detection frequency of PFNA in the serum (collected in 2009 – 2016)of mothers enrolled in the Northern California CHARGE (CHildhood Autism
Risk from Genetics and Environment) case-control study (Kim et al. 2020).

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Table 1.4 PFDA serum concentrations (ng/ml) in studies of California residents.
SampleYear

Geometric mean(ng/ml)

95%
Lower
CI

95%
Upper CI

California Teachers
Study (CTS)

2011

0.22

0.21

0.23

175
9

94.70%

Firefighter Occupational
Exposures (FOX) Project2010 to
2011

0.899

0.783

1.03

101

100%

Measuring Analytes inMaternal Archived
Samples (MAMAS)

2012 to
2015

0.198

0.174

0.226

200

83%

Biomonitoring
Exposures Study (BEST)- 1.Pilot

2011 to
2012

0.245

0.216

0.278

110

100%

Biomonitoring
Exposures Study (BEST)- 2.Expanded

2013

0.188

0.173

0.205

337

82.50%

Asian/Pacific Islander
Community Exposures(ACE) Project - ACE 1

2016

0.477

0.406

0.559

96

80.20%

Asian/Pacific Islander
Community Exposures(ACE) Project - ACE 2

2017

0.559

0.49

0.636

99

87.90%

California Regional
Exposure Study, Los
Angeles County (CARELA)2018

0.09670.08940.105

425

69.20%

California Regional
Exposure Study, Region
2 (CARE-2)

2019

0.08350.07760.0898358

65.9%

Project

N

Detection
Frequency

Data available at https://biomonitoring.ca.gov/. (Biomonitoring California 2020). Abbreviations: CI:
confidence interval; N: number of participants

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Recent findings from other human biomonitoring studies of PFDA include the following:•••

reported by NHANES for the years 2009 to 2010 reported serum
concentration levels (ng/ml; geometric mean and 95% CI) in males aged 20
years or older of 0.30 (0.28, 0.34) (Dobraca et al. 2015).
NHANES for the years 2013 to 2014 reported serum concentration levels(ng/ml; unadjusted geometric mean and 95% CI) in children aged 3–11 yearsof 0.09 (0.08 − 0.1) (Jain 2018).
A 68% detection frequency of PFDA in the serum (collected in 2009 – 2016)of mothers enrolled in the Northern California CHARGE case-control study(Kim et al. 2020).

1.3 Reviews by other health agencies
The US EPA, the National Institute for Occupational Safety and Health (NIOSH), theFood and drug Administration (FDA), and the National Toxicology Program (NTP) have
not reached conclusions or classified PFNA or PFDA as to their potential to cause male
reproductive toxicity. However, the NTP has conducted toxicity studies on PFNA andPFDA administered by gavage to Sprauge Dawley rats, reporting findings relevant tomale reproductive toxicity for both chemicals (NTP 2019). Findings from these NTP
studies are presented in sections 4 (PFNA) and 5 (PFDA) of this document.
The European Chemicals Agency (ECHA) has classified PFNA and PFDA as to theirpotential to cause reproductive toxicity (ECHA 2014, 2015). In the case of PFNA,
ECHA states that the “RAC [Committee for Risk Assessment] is of the opinion thatclassification of PFNA, PFN-S [the sodium salt of PFNA] and PFN-A [the ammonium
salt of PFNA] as Repr. [Reproductive toxicity] 2, H361f (Suspected of damaging fertility)
is warranted.” (ECHA 2014). In addition, ECHA classified PFNA and its sodum andammonium salts as Repr. 1B, H360Df (May damage the unborn child)” and “Lact.
[Lactation] H362 (May cause harm to breast-fed children),” based on some data on the
reproductive toxicity of PFNA and on read-across from perfluoroocanotic acid (PFOA)(ECHA 2014).
In the case of PFDA, ECHA stated “In conclusion, RAC agrees with DS [dossier
submitter] that overall, the data provide some evidence of adverse effects on sexualfunction and fertility for PFDA and that classification of PFDA as Repr. 2; H361f(Suspected of damaging fertility) is warranted” (ECHA 2015). In addition, ECHA
classified PFDA as “Repr. 1B; H360Df (May damage the unborn child)” and “Lact.
[Lactation] H362 (May cause harm to breast-fed children),” based mostly on data fromPFOA (ECHA 2015).

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1.4 Overview of systematic literature review approach
Searches of the published scientific literature on the developmental and reproductive
toxicity (DART) of PFNA and PFDA (and their salts) were conducted in February 2021.
The searches sought to identify peer-reviewed open source and proprietary journal
articles, print and digital books, reports and gray literature that potentially reported
relevant toxicological and epidemiological information on the developmental andreproductive toxicity of these chemicals.
Three types of searches were conducted:•••

Primary searches in major biomedical databases, conducted by OEHHA librarian
Nancy Firchow, MLS
Searches in other data sources, including authoritative reviews and reports, anddatabases or web resources, conducted by OEHHA scientists
Additional focused searches, conducted by OEHHA scientists
Search process

The US EPA Computational Toxicology (CompTox) Chemicals Dashboard(https://comptox.epa.gov/dashboard) was used to identify synonyms for PFNA andPFDA (and their salts). The PubMed MeSH database(https://www.ncbi.nlm.nih.gov/mesh/) was used to identify subject headings and otherindex terms related to the chemicals, reproduction and development, and adverse
effects on reproduction and development.
Preliminary searches were run and results evaluated to identify additional relevant
search terms. The resulting search strategies were then executed in PubMed twice foreach chemical (and its salts), limiting the first search to human studies, and the second
search to non-human studies. There were no restrictions in the searches on exposure
route or duration of exposure, or on publication language. The full DART search strings
used in PubMed are included in Appendix A.
The PubMed search strategies were then tailored for use in the additional databases
and data sources listed below, according to the search interface and features unique toeach resource. For instance, MeSH terms were replaced with Emtree terms for the
Embase search strategies.
Data sources:
The following is a list of the major data sources (biomedical literature databases)
searched to find information on PFNA and its salts and PFDA and its salts.
•

PubMed (National Library of Medicine) (https://www.ncbi.nlm.nih.gov/pubmed)

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•••

Embase (https://www.embase.com)
Scopus (https://www.scopus.com)
SciFinder-n (https://scifinder-n.cas.org/)

In addition to the systematic literature searches, OEHHA asked the public to identify
pertinent references through a data call-in that was open from March 26, 2021 to May
10, 2021. No references were submitted for consideration.
Literature screening process
The results of these literature searches were uploaded to EndNote libraries (human andnon-human [i.e., experimental animal and cell-free] results were kept separate) andduplicates were removed. A total of 652 and 472 references were identified for PFNA
and its salts and for PFDA and its salts, respectively, through this initial literature search
process. In addition to the studies identified through this process, other relevant studies
were identified from citations in individual articles, and through alert services (e.g.,
ScienceDirect, Google Scholar, etc.).
The EndNote libraries containing the literature search results (citations) for PFNA andits salts and PFDA and its salts were uploaded to two separate HAWC (Health
Assessment Workspace Collaborative, https://hawcproject.org) projects. HAWC is atool used for multi-level screening of literature search results.
In Level 1 screening, citations were reviewed independently by OEHHA scientists,based solely on study titles and abstracts, using specific inclusion and exclusion criteria
to eliminate studies or articles that did not contain information on DART or other key
related topics (e.g., pharmacokinetics, mechanisms of action). This initial screen (Level1) was intended to identify all studies deemed to have a reasonable possibility ofcontaining information relevant to DART that could be useful for the review process, andto further identify (i.e., tag in HAWC) studies relevant to particular aspects of DART
(e.g., male reproductive toxicity, female reproductive toxicity, developmental toxicity).
For purposes of identifying the available evidence on the male reproductive toxicity ofthese chemicals, citations identified as having a reasonable possibility of containing
information relevant to male reproductive toxicity underwent Level 2 screening. In theLevel 2 screening of this subset of citations, the full text was obtained. These full
papers were screened independently by one OEHHA scientist, using similar
inclusion/exclusion criteria as was used in the Level 1 screening. However, Level 2
reviewers could make more accurate judgements about the relevance of the articles
because they were reviewing the full text in addition to the title and abstract. Following
Level 2 screening, the tagging of articles according to key topics was updated in HAWC.
Level 1 and 2 screenings were repeated as search results were updated, and with

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additional relevant studies identified from citations in individual articles and alert
services (e.g., ScienceDirect, Google Scholar). (See Appendix A for additional details).
Literature searches were last updated in July 2021. One hundred and fourteen
references were cited in this document.

2. PHARMACOKINETICS OF PFNA AND PFDA
2.1 Absorption
In general, PFASs can be absorbed following either oral, inhalation, or dermal
exposures. The principal route of exposure in humans is the oral one followed byinhalation and dermal (Poothong et al. 2020). Studies conducted in animals indicatethat about 95% of PFNA and PFDA are absorbed after oral administration (ATSDR2021). These findings on oral absorption are similar to those for PFOA (92%) andperfluorooctane sulfonic acid (PFOS) (approximately 98%) in gavage studies in malerats (Cui et al. 2010). No quantitative estimates of the fractional absorption of PFDA or
PFNA following inhalation or dermal exposure were identified (ATSDR 2021).
It is expected that PFNA and PFDA salts readily dissociate in aqueous media to form
the corresponding organic acid, similar to observations that ammonium
perfluorooctanoic acid in the presence of water freely forms PFOA (Hundley et al.
2006).
2.2 Distribution
In general, PFASs are present in the blood, and widely distributed in tissues throughout
the body. A study measuring a number of PFASs in various human tissues (i.e., liver,kidney, brain, lung, and bone [rib]) reported the highest levels of PFNA in brain andkidney, with lower levels in lung and liver, and no detectable PFNA in bone (Pérez et al.
2013). In this same study the highest levels of PFDA were reported in brain, followed
by lung and then kidney, with no detectable PFDA in liver or bone.
In studies in laboratory animals, the highest concentrations of PFASs are generally
found in the liver, kidneys, and blood (ATSDR 2021). For example, in studies in maleSprague Dawley (SD) and Wistar rats the order from highest to lowest for maximum
concentration (Cmax) in various tissues following a single gavage dose of PFNA wasliver> kidney> serum> lungs> heart> spleen> testes> muscle> fat> intestines> brain(Benskin et al. 2009; Iwabuchi et al. 2017). For PFDA, in studies in male Wistar rats
after a single gavage dose, the PFDA tissue distribution was about 86% in liver; 13% inserum and 1.4% in brain (Kawabata et al. 2017; Kudo and Kawashima 2003). Wistar
rats administered a single intraperitoneal injection of PFDA (20 mg/kg) had a greater
proportion of the PFDA in serum (10%) than liver (5%) when assessed seven days after
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injection (Ylinen and Auriola 1990). Covalent binding of PFDA was detected in plasma,
liver and testes, with a higher relative concentration of bound PFDA in the testis
compared to plasma or liver (Vanden Heuvel et al. 1992).
Information on the tissue distribution of PFNA, PFDA, and PFASs is also available fromstudies in fish, chicks, and the Artic fox. In one study of PFNA in male and female
zebrafish, levels were reported to be two times higher in male gonads than in female
gonads (Zhang et al. 2016). In a study conducted with one-day-old male chickens
exposed orally by gavage to a mixture of three PFASs (including PFDA) for three
weeks, the highest levels of PFDA were in the liver, followed by kidney and blood(Yeung et al. 2009). In studies in rainbow trout that measured tissue PFAS
concentrations, the highest levels were in liver, followed by blood, kidney, and skin (Falket al. 2015; Goeritz et al. 2013). And in a study in Artic foxes, blood and liverconcentrations of PFNA and PFDA were inversely correlated with levels of body fat,suggesting that body fat composition may be a factor in the tissue distribution andaccumulation (Aas et al. 2014).
Transport
PFASs appear to bind to proteins in blood. PFNA and PFDA are primarily transportedby albumin in humans (Forsthuber et al. 2020). In an in vitro equilibrium dialysis study,
more than 99.9% of PFNA was bound to bovine or human serum albumin (Bischel et al.
2010). In rats PFDA was shown to be bound to serum proteins (Ylinen and Auriola1990). Covalent binding of PFDA to proteins in plasma was observed in rats following
in vivo administration of radiolabeled [1-14C]PFDA (Vanden Heuvel et al. 1992). In in
vitro studies these authors showed that PFDA covalently binds to rat albumin andhemoglobin, and that covalent binding of PFDA to albumin was time- and dosedependent (Vanden Heuvel et al. 1992).
Maternal-fetal transfer
PFASs can be transferred to the human fetus during pregnancy (ATSDR 2021). PFASs
were found in the serum of pregnant women and in fetal cord serum in Korean, Chineseand Spanish populations (S-K Kim et al. 2011; S Kim et al. 2011; J Liu et al. 2011;
Manzano-Salgado et al. 2015). Several PFASs have been detected in maternal serum,in the placenta and in embryos or fetal tissues. In a Danish population PFNA and PFDA
were detected throughout pregnancy in maternal serum samples collected between
2014 and 2015, with a frequency similar to that of PFOA and PFOS; the relative
frequency of detection was PFOA>PFOS>PFNA>PFDA (Mamsen et al. 2019). In asmall study (N = 32) of placentas collected in a Chinese population in 2010, placental
concentrations of PFNA and PFDA were eight times lower than concentrations of PFOSPFNA and its salts andPFDA and its salts

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(T Zhang et al. 2013). PFNA and PFDA appear in fetal liver and lungs with increasing
frequency from the second trimester, with PFNA having a higher detection frequency
than PFDA (Mamsen et al. 2019).
Several studies have reported that PFNA concentrations in maternal serum are higher
than in cord serum (Manzano-Salgado et al. 2015; Needham et al. 2011; T Zhang et al.
2013), and this has also been reported for PFDA (T Zhang et al. 2013). In a recent
study in matched maternal−cord serum pairs assessing the transplacental transfer
efficiencies of PFNA and PFDA, lower transfer efficiencies were observed in pretermcompared to full-term deliveries, with statistically significant lower PFNA and PFDA
levels in preterm compared to full-term cord serum (p<0.001) (Jing Li et al. 2020).
Breast milk transfer
PFASs can be transferred to nursing infants via breastmilk (ATSDR 2021). PFNA wasdetected in 100% and PFDA in 78% of milk samples collected from a population ofnursing mothers in China (J Liu et al. 2011). However, PFNA and PFDA levels in breast
milk were below the limit of detection in a study conducted in a Korean population (S-K
Kim et al. 2011; S Kim et al. 2011) and in a cohort from the Faroe Islands (Needham etal. 2011).
Sex differences in distribution
Studies in humans and animals suggest that there may be differences between males
and females in the distribution within the body of PFNA and PFDA. In humans, slightly
higher ratios of mean cord to maternal serum concentrations were observed in boys
compared to girls for both PFNA and PFDA, although the differences did not reachstatistical significance (J Liu et al. 2011). In rats, the concentration of PFNA in the liver
was dependent on exposure dose and sex (Kudo and Kawashima 2003). PFNA
accumulation in rat liver was greater in males than in females administered PFNA byintraperitoneal injection (Kudo et al. 2000; Kudo et al. 2001; Kudo and Kawashima2003), and the concentration of PFNA in the serum was also higher in males thanfemales after a single intraperitoneal exposure (Kudo et al. 2001). This higher
accumulation of PFNA in the liver of male rats was dependent on testosterone (T), as
liver concentrations were reduced in castrated male rats, and supplementation of
castrated males treated with exogenous testosterone had PFNA liver concentrations
comparable with those seen in intact males (Kudo et al. 2000; Kudo and Kawashima2003). For PFDA, no differences in the levels of PFDA in rat liver were observed
between males and females, following intraperitoneal injection (Kudo et al. 2000; Kudo
et al. 2001; Kudo and Kawashima 2003), while females had higher PFDA serum levels

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than males following intraperitoneal injection (Kudo et al. 2001) and higher PFDA
plasma levels than males following intravenous injection (Dzierlenga et al. 2020).
2.3 Metabolism
Several studies for oral and intraperitoneal exposure as well as in vitro studies suggestthat PFASs including PFDA are not metabolized and do not undergo chemical reactionsin the body (ATSDR 2021; Ylinen and Auriola 1990). The ATSDR review reported theabsence of studies examining metabolism of PFASs following inhalation or dermal
exposure then they conclude that “metabolism by these exposure routes is notexpected” (ATSDR 2021).
2.4 Excretion
Excretion pathways of PFNA and PFDA include urinary and fecal excretion, and for
women, pregnancy (e.g., transfer to fetus), and lactation. PFNA and PFDA may also be
eliminated from the body through incorporation into (finger and toe) nails.
In a small biomonitoring study of individuals in China with no known occupational
exposures to PFNA or PFDA (N=39), higher detection frequencies of PFNA and PFDA
were observed in the nails of study participants (51.2% and 56.4%, respectively) than in
the urine (5.13% and 7.7%, respectively) (Wang et al. 2018).
Studies in humans, mice and rats indicate that both PFNA and PFDA are slowly
eliminated from the body (Fujii et al. 2015; Kudo et al. 2001). Studies of humans andmice have found that PFNA is excreted to a slightly greater extent in urine compared tofeces, while the opposite is the case for PFDA (Fujii et al. 2015). Studies in rats indicatethat both PFNA and PFDA are excreted to a greater extent in feces compared to urine(Kudo et al. 2000; Kudo et al. 2001; Vanden Heuvel et al. 1991).
2.5 Species differences in PFNA and PFDA serum half-lives
PFASs, including PFNA and PFDA, demonstrate species differences in half-life, withestimates for serum half-lives of longer-chain PFASs, including PFNA and PFDA, on theorder of days for rodents, and years for humans.
PFNA
In a human study (N=86 paired blood and morning urine samples) from healthy
volunteers from two cities in China (Shijiazhuang, Handan), the geometric mean serum
half-life for PFNA was estimated to be 3.2 years in men (Y Zhang et al. 2013). In male
Wistar rats exposed to 22 mg/kg of PFNA by the intravenous route, the half-life was 30days (NTP 2019). In studies of male SD rats, the plasma half-life for PFNA was 40 days
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following intravenous exposure to 3 mg/kg, and ranged from 30 to 55 days following oral
exposures (i.e., 10-14 µg/day via diet for 12 days; 3 mg/kg via gavage) (De Silva et al.
2009; Kim et al. 2019).
PFDA
In the same human study described above for PFNA, it was estimated that thegeometric mean serum half-life for PFDA was 7.1 years in men (Y Zhang et al. 2013).
The serum half-life for PFDA has been investigated in two studies of male SD rats, andestimates ranged from 36 to 109 days following intravenous exposure to 1 or 2 mg/kg
PFDA, and from 68 to 80 days following oral exposures to 1 or 2 mg/kg PFDA
(Dzierlenga et al. 2020; Kim et al. 2019).

3. ENDOCRINE SYSTEM INVOLVEMENT IN DEVELOPMENT AND
FUNCTION OF THE MALE REPRODUCTIVE SYSTEM
Reproductive biology is under close control from the endocrine system. In addition to
the intrinsic regulation present in the organism, environmental factors may influence theendocrine system and the reproductive processes controlled by it. Effects ofenvironmental exposures on the endocrine system can occur through a variety ofmechanisms, including alterations in hormone synthesis or metabolism, hormone
transport and binding to proteins in blood, and target cell receptor mediated events,
including transactivation and intracellular signaling. Studies investigating the effect of
PFNA or PFDA on aspects of the endocrine system that may be relevant to thedevelopment or function of the male reproductive system are discussed in sections 4(PFNA) and 5 (PFDA). Here we provide a brief overview of the role of the
hypothalamus-pituitary-gonad axis and the thyroid hormone system in male
reproductive system development and function.
3.1 Hypothalamus-pituitary-gonad axis
The hypothalamus-pituitary-gonad (HPG) axis controls the function of the testes throughgonadotropin releasing hormone (GnRH), which is produced in the hypothalamus, andthe gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH),which are produced in the pituitary.
The endocrine function of the testes starts early in life, at sexual differentiation during
fetal development. Early stages of sexual differentiation involve regression of the
Müllerian duct (precursor of the female internal reproductive tract) by action of
antimüllerian hormone (AMH), which is produced by fetal testicular Sertoli cells.

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Sex steroids also play an important role in male sexual differentiation, as testosterone(produced in the testes and dihydrotestosterone (DHT, produced in the brain, testes,and other organs), the most potent of the androgens, act together in the virilization of
the male fetus. In adults, the testes are responsible for producing not only germ cells
but also hormones that are important for normal reproductive function. Under the
regulatory control of LH, androgens are produced in Leydig cells by a series of
enzymatic reactions starting from cholesterol. Secreted testosterone can be further
metabolized, primarily in peripheral tissues, to form either DHT or estradiol (E2).
Spermatogenesis requires healthy Sertoli cells and appropriate hormonal control. FSH
and testosterone act on specific Sertoli cell receptors (located on the plasma membraneor intracellularly, respectively) in a coordinated manner to facilitate normal sperm
production.
There is evidence in animal models that PFNA and PFDA are acting as endocrine
disrupting chemicals, i.e., affecting hormone production, metabolism, and receptor
mediated effects. Certain aspects of HPG axis disruption have been investigated for
PFNA and PFDA, and will be discussed in subsequent sections.
3.2 Thyroid hormones
Thyroid hormones regulate basal metabolic rate, as well as exerting control overgrowth, development, and differentiation of many cells and organ systems — including
the testes (as reviewed by (Rajender et al. 2011)). Despite some inconsistencies amongstudies, as well as species differences, reviews of thyroid hormones in male
reproduction and infertility found indications that short-term hypothyroidism in postpubertal males can have adverse effects on sperm motility, semen volume, and semenquality (Alahmar et al. 2019; Krajewska-Kulak and Sengupta 2013). The thyroid
hormones, tri-iodo-L-thyronine (T3 or triiodothyronine) and tetra-iodo-L-thyronine (T4 or
thyroxine), are produced and secreted by the thyroid gland in response to the regulatory
hormones thyroid stimulating hormone (TSH) from the pituitary gland, and thyrotropinreleasing hormone (TRH) from the hypothalamus (Chang et al. 2008; Rajender et al.
2011). Once secreted, T4 can be converted to T3 or to reverse triiodothyronine (rT3, an
inactive isomer of T3) (Chang et al. 2008; Gutshall et al. 1989).
Thyroid hormone receptors (TRs) have been identified on testicular cells, and T3 binds
directly to TRs on Sertoli cells. Binding to Sertoli cell TRs activates gene transcription
and protein synthesis, as well as Sertoli cell proliferation and differentiation (Alahmar etal. 2019; Rajender et al. 2011). The binding of T3 to Sertoli cell TRs is suspected tohave a role in initiating sperm development. While the Rajender et al. (2011) review
notes the existence of contradictory reports as to how thyroid hormone acts on Leydig
as well as Sertoli cells, proposed mechanisms suggest a role for T3 in stimulating basal
testosterone generation.
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Disruption of thyroid homeostasis associated with exposure to PFASs, including PFDA
and PFNA, has been reviewed (Xie et al. 2020) and attributed to several potential
mechanisms including:••••Competitive displacement of T4 from binding to thyroid hormone transport
proteins.
Activation of TRs and other nuclear receptors.
Effects on expression of genes related to thyroid hormone signaling.
Regulation of enzymatic activities in the thyroid gland.

Experimental treatment of rats with PFOS, for example, was not found to alter thyroid
gland histology or produce elevated TSH consistent with primary hypothyroidism(Chang et al. 2008). Observed lowering of serum total thyroxine with PFOS treatment
was not attributable to disruption of the hypothalamic-pituitary-thyroid (HPT) axis orinterference with release of TSH. The authors suggested that PFOS may compete with
T4 for binding with transport proteins, leading to increased turnover and elimination, inturn resulting in reduced serum total T4.

4. PFNA AND ITS SALTS: MALE REPRODUCTIVE TOXICITY
4.1 PFNA: Human studies of male reproductive effects
OEHHA identified 17 epidemiologic studies of possible effects of PFNA on the male
reproductive system. These studies examined one or more of the following types ofoutcomes: anogenital distance (AGD); outcomes related to male reproductive function,
including reproductive hormones, semen quality parameters, DNA damage to sperm,
and in vitro fertilization (IVF) outcomes; and cancer. The studies are ordered byoutcomes, and within outcomes, by publication date, in the following discussion and in
Table 4.1. Some studies included multiple outcomes but are included only once in thediscussion and table. Statistically significant results in Table 4.1 are in bold type. In
the following summary, all results are statistically significant unless otherwise noted.
Anogenital distanceTwo prospective cohort studies examined associations between prenatal PFNA
exposure and anoscrotal (AGDAS) and anopenile (AGDAP) distance, reporting conflicting
findings. Tian et al. (2019), with higher maternal plasma PFNA concentrations (median= 1.75, interquartile range (IQR) = 1.30, 2.47) and larger sample size (n=500), reported
non-statistically significant decreases in AGDAS and AGDAP at birth that disappeared by
the age of 12 months. In the study by Lind et al. (2017), maternal serum PFNA
concentrations were lower: median = 0.7, IQR = 0.5, 0.9 ng/mL. Lind et al. reported an
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increase in AGDAS at three months, and no associations with AGDAP (Lind et al. 2017;
Tian et al. 2019).
Male reproductive function
Twelve studies examined male reproductive function, including reproductive hormone
levels, semen quality parameters, and IVF outcomes, in association with PFNA. Of theseven studies reporting results for associations between PFNA exposure and T, tworeported decreases in T for adult men (Cui et al. 2020) and adolescent boys (Zhou et al.
2016), a third reported a nonsignificant decrease in total T in young men (Joensen et al.
2013), three studies reported no associations (Joensen et al. 2009; Joensen et al. 2013;
Lewis et al. 2015; Ma et al. 2021) and one reported inconsistent evidence acrosslocations (Specht et al. 2012). For E2, three of four studies reported no associations(Cui et al. 2020; Ma et al. 2021; Specht et al. 2012) and one reported a decrease
associated with PFNA (Joensen et al. 2013). No consistent pattern of associations isapparent for other reproductive hormones. Cui et al. (2020) reported decreased sex
hormone-binding globulin (SHBG) associated with both semen and serum PFNA and alower T/LH ratio associated with semen PFNA (Cui et al. 2020). The data suggest that
PFNA may be associated with lower T, but not other reproductive hormones and related
proteins.
Evidence for associations of PFNA with the percentage of sperm with normal
morphology and progressive motility was mixed: Louis et al. 2015 reported an increasein the percentage of normal sperm and a decrease in the percentage with coiled tails,
three studies reported non-significant associations with lower proportions ofmorphologically normal sperm (Ma et al. 2021; Pan et al. 2019; Toft et al. 2012), andone reported no associations (Joensen et al. 2013). Evidence for associations between
PFNA and sperm viability, curvilinear velocity (VCL), and straight line velocity (VSL) was
sparse (Louis et al. 2015; Pan et al. 2019). Of the six studies that examined effects of
PFNA exposure on sperm concentration and count, five reported no associations(Joensen et al. 2013; Louis et al. 2015; Pan et al. 2019; Petersen et al. 2018; Toft et al.
2012), while a study of a cohort of couples undergoing IVF reported that PFNA is
associated with a substantial reduction in sperm concentration and a non-statisticallysignificant reduction in sperm count (Ma et al. 2021). IVF outcomes, however, weregenerally not associated with PFNA (Ma et al. 2021).
Three studies examined possible effects of PFNA on sperm DNA. Specht et al. (2012)
reported no associations with sperm DNA fragmentation using the sperm chromatin
structure assay (SCSA) and the in situ terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assays. However, Pan et al. (2019) also conducted the SCSA
and reported that semen PFNA was associated with small increases in DNA
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fragmentation index (DFI), and both semen and serum PFNA were associated withsmall increases in high DNA stainability (HDS) indicative of damage to integrity of DNA(Pan et al. 2019). DNA methylation parameters were generally not associated with
PFNA (Leter et al. 2014).
CancerTwo studies examined associations between PFNA and prostate cancer or prostatespecific antigen (PSA) level. A case-control study in Sweden reported that PFNA
exposure above the control median was not associated with prostate cancer (Hardell et
al. 2014). In a sample from the C8 Study, PFNA was not associated with PSA level(Ducatman et al. 2015).
A case-control study in Shanghai, China investigating the association between serum
PFAS levels and germ cell tumors in children did not report findings by tumor location(e.g., testes vs. ovaries) or sex of the child (Lin et al. 2020). Because of theselimitations in reporting, the study is uninformative for purposes of evaluating male
reproductive system hazards, and the study was not included in Table 4.1.

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Table 4.1 PFNA: Epidemiologic studies of male reproductive toxicity 2.
Reference; study
location, period,
design, sample
Lind et al., 2017
Denmark
2010-2012
Prospective cohort

Exposure matrix
and concentration(ng/mL)
Maternal serum collected
at gestational week 5-12
(median 10 weeks)
Median (IQR) 0.7 (0.5-0.9)

Outcomes
AGDAS & AGDAP andpenile width at 3
months, measured
three times by trained
study technicians

N=299

Shanghai, China
2012
Prospective cohort
N=500 male singletons born
to women who wererecruited at gestational
weeks 12-16 from one
hospital

AGDAS
β (95% CI) per unit increase in ln(PFNA): 1.4 (0.02,
2.9) mm
No association when 2nd, 3rd, 4th quartiles of PFNA
were compared to 1st
AGDAP, penile width: no associations

Odense Child Cohort: sonsof mothers who were >16
years old, of Western origin,
had no communication
barriers, delivered in areference hospital, and did
not use reproductive
assistance.
Tian et al., 2019

Results

Covariates, other
PFASs analyzed
Adjusted for “post-conceptionalage” (sum of GA at birth and ageof child at the AGD
measurement, in days), z-score
for weight at 3 months, parity,maternal smoking, and prepregnancy body mass index(BMI)
Other PFASs:
perfluorohexane sulfonic acid
(PFHxS), PFOS, PFDA

Maternal plasma (fasting)collected at 12-16 weeks
gestationMedian (5, 25, 75, 95
percentiles): 1.75 (0.73,
1.30, 2.47, 3.99)
Geometric mean (GA;standard deviation or SD):
1.8 (1.68)

AGD [AGDAS &AGDAP] at birth, 6months, and 12
monthsMeasured by trained
examiners with noknowledge of maternal
PFAS concentrationsβ (95% CI) for unit change in ln(PFNA) (mm)AGDAS
Birth: –0.51 (–1.19, 0.17)
6 months: –1.83 (–4.12, 0.45)
12 months: no association
AGDAP
Birth: –0.34 (–1.14, 0.46)
6 months: –0.87 (–3.17, 1.44)
12 months: no association
In longitudinal analyses, no associations with AGD

Selected a priori and adjusted
for: maternal age, education,
parity, and pre-pregnancy BMI;
GA at birth; age at examination(days), infant size (weight atbirth, weight-for-length z-score at
6 and 12 months of age)
Stratified by breastfeeding
duration for AGD at 6 and 12
months8 PFASs detected in ≥80% ofparticipants: PFHxS, PFOA,PFOS, PFNA, PFDA, PFUnDA,
PFDoA, PFTrDA

Comments
Correlations among PFASs
not reported.
Women who gave birth in2010 had higher PFOA,PFOS, and PFNA
concentrations than women
who gave birth in 2011-2012.
Women who participated inthis study were better
educated, smoked less, andwere more likely to be ofDanish origin than
nonparticipants.
Pearson correlation coefficient
for ln-transformed PFNA and:PFOS r = 0.64
PFOA r = 0.39
PFDA r = 0.79
PFUnDA r = 0.796
PFDoA r = 0.47
PFTrDA r = 0.31
75% of mothers had
university-level education or
higherStudies are ordered by outcomes (AGD, male reproductive function, prostate cancer), and within outcomes, by publication date. Statisticallysignificant results are in bold type.
2

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Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Joensen et al., 2009

Serum

DenmarkMedian (5th, 95th
percentiles)

2003
Cross-sectional*
N=105
Healthy young men from thegeneral population reportingfor the military draft, medianage 19 years.

All 0.8 (0.4, 1.8)
High T 0.8 (0.4, 2.0)
Low T 0.8 (0.4, 2.0)

Outcomes
Serum reproductive
hormones and related
proteins: T, E2, sex
hormone-binding
globulin (SHBG), LH,
follicle-stimulating
hormone (FSH),
inhibin B, free
androgen index (FAI)

Results
No associations with T
Due to low concentration, PFNA was not includedin further analyses

Covariates, other
PFASs analyzed

Comments

PFNA was included only inbivariate analyses (noadjustments)
Other PFASs: PFHxS, PFPA,PFOA, PFOS, PFOSA, PFDA,
PFUnDa, PFDoA, PFTrA

Semen quality: semen
volume; sperm
concentration, count,% motile, morphology*Men with highest andlowest T levels wereselected.
Group 1, n=53, median 31.8
nmol/LGroup 2, n=52, median 14.0
nmol/LSpecht et al., 2012

Serum

Kharkiv, Ukraine; Greenland;
Warsaw, Poland

Median (range)

2002-2004

Greenland 1.4 (0.5, 12)

Cross-sectional
N=604

Ukraine 1.0 (0.2, 4)
Poland 1.2 (0.5, 6)

Men who provided semen
and blood samples andcompleted questionnaires,and were partners ofpregnant women enrolled in
the INUENDO fertility cohort

Percentage of spermwith detectable DNA
fragmentation by
SCSA
DNA fragmentation byin situ TUNEL assay
Pro- (Fas) and antiapoptotic (Bcl-xL)
markers on sperm
Serum: FSH, LH, E2,
T, SHBG

No associations with sperm DNA fragmentation(SCSA and TUNEL assays) (data not reported).
No consistent associations with apoptotic markers
across study locations or models within locations(data not reported).
No consistent relationship with SHBG.
Associations with T, E2, and the gonadotropins
FSH and LH were not consistent across locations(data not reported).
Results for inhibin-B were not reported

Analyses were stratified by study
location and adjusted for sexual
abstinence period, age, BMI,
caffeine consumption, serum
cotinine, recent fever, selfreported genital infections,
testicular disorders, and semen
spillage.
Alcohol use was not included
due to frequency of missing
data.
Other PFASs: PFHxS, PFOA,
PFOS

Inhibin B

Spearman’s rank correlations
among PFASs ranged from
r=0.4 to r=0.9, thoughcorrelations with PFNA were notreported.

PFNA and its salts andPFDA and its salts

20

All men had pregnant
partners, thus sterile andhighly subfertile men are
underrepresented.
Men were asked to collectsemen after ≥2 days ofabstinence
High frequency of missing
data for Bcl-xL and Fas due tosamples lost during shipment
and insufficient number ofcells for analysis.
Many reproductive hormone
samples were missing forGreenland (32%) and Poland(41%).

OEHHA
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Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Toft et al., 2012

Serum median (33rd, 67th
percentiles)

Kharkiv, Ukraine; Greenland;
Warsaw, Poland

All 1.2 (1.0, 1.5)

2002-2004

Greenland 1.7 (1.3, 2.4)

Cross-sectional

Poland 1.2 (1.0, 1.3)

Ukraine 1.0 (0.8, 1.2)

Outcomes

Results

Semen quality: sperm
concentration, volume,
total count, %morphologicallynormal sperm, %motile sperm

No associations with sperm concentration, volume,
total count, % motile sperm (data not reported)
NS association with lower % of normal sperm when
PFNA was analyzed as a continuous variable (datanot reported)

N=588

Comments

Selected a priori and adjusted
for: age, abstinence time, semen
spillage, current smoking, historyof urogenital infections, BMI,
country (combined analyses)

Participation rates:

Sperm motility analyses wererestricted to samples analyzed
within 1 hour of collection

All men had pregnant
partners, thus sterile andhighly subfertile men are
underrepresented.

Other PFASs: PFHxS, PFOA,
PFOS

Men who provided semen
and blood samples andcompleted questionnaires,and were partners ofpregnant women enrolled in
the INUENDO fertility cohort

PFNA and its salts andPFDA and its salts

Covariates, other
PFASs analyzed

Kharkiv 36%
Greenland 79%
Warsaw 29%

Men were asked to collectsemen after ≥2 days ofabstinence
Volume and total sperm countanalyses were restricted tosamples with no reported
semen spillage

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Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Joensen et al., 2013

Serum

DenmarkMedian (IQR) 1.07 (0.88,
1.41)

2008-2009

Mean ± SD 1.23 ± 0.63

Cross-sectional
N=247
Healthy young men
randomly selected from thegeneral population, medianage 19 years

Outcomes
Serum reproductive
hormones and related
proteins: total T, freeT, E2, inhibin B, FAI;
T×100/SHBG), FSH,
LH, SHBG
Semen quality:
volume; sperm count,concentration,
motility, morphology,
total normal sperm

Resultsβ (CI) per ng/mL increase in PFNA
Hormones (ln-transformed)
Total T β = –0.059 (–0.118, 0.001) nmol/L

Covariates, other
PFASs analyzed
Analyses with hormones and
SHBG were adjusted for BMI
and smoking

E2 β = –0.075 (–0.013, –0.019) pmol/L

Semen volume, concentration,and total count were adjusted for
abstinence time

No associations with other reproductive hormones
or SHBG% morphologically normal sperm
was unadjusted

Semen quality and testicular volume: noassociations

Progressively motile % wasadjusted for time to semen
analysis

Free T β = –0.052 (–0.114, 0.010) nmol/L

Testicular volume

Considered but not included:
time of day of blood sample,
ethnicity, recent alcohol use,prenatal exposure to tobaccosmoke, previous or current
diseases, recent fever, recent
medication use, season,interaction with cigarettesmoking

Comments
Participation rate was ~30%,
which is “higher than otherpopulation-based semenquality studies”.
Participants were recruited at
a compulsory medical
examination for consideration
for military service
Men were asked to abstain for
48 hours; median abstinence
period was 62 hours.
Analyses were blinded

Other PFASs: PFHxS, PFHpS,PFOA, PFOS, PFDAPFNA and its salts andPFDA and its salts

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Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Leter et al., 2014

Serum

Kharkiv, Ukraine; Greenland;
Warsaw, Poland

Mean ± SE:

2002-2004

Greenland 2.2 ± 0.2

Cross-sectional
N = 262 from 607 malepartners of pregnant women
enrolled in the INUENDO
fertility cohort (312 samples
were randomly selected fromthose with sufficient semen.
Further sample loss is due to“random unpredictable cellloss occurring during theprocessing of somesamples.”

Kharkiv 1.1 ± 0.1
Warsaw 1.4 ± 0.1
Combined 1.6 ± 0.1

United StatesMedian (IQR) by age
group:

N = 857 males, 12-80 years
Subset of the NHANES withdata on serum PFASs, T,
and thyroid hormones

Results were inconsistent across study location
β (CI) per unit ln(PFNA):Alu
For each study location, the association was
negative but ns
LINE-1
Kharkiv β = 5.7 (1.4, 10.1) %No associations for other study locations
SatαKharkiv β = 9.3 (1.5, 17.1) %Covariates, other
PFASs analyzed
Adjusted for age, smoking(selected a priori), study location
combined analyses)
Other PFASs and correlations:
PFHxS r=0.556
PFOA r=0.417
PFOS r=0.571

Comments
All men had pregnant
partners, thus sterile andhighly subfertile men are
underrepresented.
Men were asked to collectsemen after ≥2 days ofabstinence

PFDA*, PFUnDA*, PFDoDA**excluded due to low detectionrate

No associations for other study locations
FCM DGML
Warsaw β = –99.6 (–152.5, –46.8) units

Serum

Cross-sectional

Sperm DNA global
methylation levels,
indicated by a)average DNA
methylation in
repetitive DNA
sequences (Alu, LINE1, Satα) and b) flow
cytometric (FCM)
immunodetection of 5methyl-cytosines using
the FCM Sperm DNA
Global Methylation(DGML) Assay,
performed by blinded
investigators

Results

No associations for other study locations

Lewis et al., 20152011-2012

Outcomes

12 to <20 years: 0.78
(0.56, 1.19)
20 to <40 years: 0.98
(0.67, 1.31)
40 to <60 years: 1.00
(0.67, 1.57)

Serum total T, thyroid
stimulating hormone(TSH), and free andtotal T and free andtotal thyroxine (T4)

Percent change (95% CI) in hormone
concentrations per doubling of PFNA:
No associations with T
12 to <20-year-olds:
TSH 16.3 (4.0, 30.2), p < 0.05
40 to 60-year-olds:
Total T4: –2.5 (–5.2, 0.2), p < 0.1.
No other associations with thyroid hormones

Adjusted for age, body massindex (BMI), poverty income
ratio, race/ethnicity, serum
cotinine

The largest association of any
PFAS and thyroid hormones
was that of PFNA with TSH in
12-20 year olds.

Other PFAS: PFHxS, PFOA,

Significant reduction ingeometric mean levels of the
included PFASs compared toprevious NHANES studies

PFOS
Correlations among PFASs were
not reported

60 to 80 years: 1.07 (0.77,
1.58)

PFNA and its salts andPFDA and its salts

23

OEHHA
October 2021

Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Outcomes

Results

Louis et al., 2015

Serum

Semen quality:

Associations with 1-unit increase in ln(PFNA):

Michigan and Texas

Median (IQR); geometricmean (95% CI)

Volume, straw
distance, sperm
concentration, total
count, hypo-osmotic
swollen,No significant differences in semen volume; sperm
viability, count, or concentration.

2005-2009
Cross-sectional

Michigan: 1.0 (0.75, 1.35);
0.96 (0.84, 1.11)

N=462

Texas: 1.65 (1.2, 2.2); 1.68
(1.61, 1.76)

Population-based sample ofcouples planning pregnancy,
recruited from a marketing
database in Michigan and afishing/hunting license
registry in Texas.

8 motility measures,
6 sperm headmeasures,12 individual and 2
summary morphology
measures,% normal sperm, strict criteria, β= 3.897 (0.564,
7.231)% of sperm with coiled tail, β= –4.030 (–7.766, –0.293)

Covariates, other
PFASs analyzed
Adjusted for age, BMI, serum
cotinine, abstinence, sample
age, research site
(Texas/Michigan)
Other PFASs: PFOA, PFOS,PFOSA, Et- PFOSA-AcOH, MePFOSA-AcOH, PFDA

Serum sampled after 8hour fast

Cross-sectional
N=102 healthy 13-15-yearold boys from 7 publicschools

Other PFASs: PFBS, PFHxA,
PFHxS, PFOA, PFOS, PFDA,
PFDoA, PFTA

2009-2010

Median (IQR) 0.8 (0.6, 1.0)

Serum T and E2(average of 2 values)

A 2nd sample collected
approximately 1 month later
was used to corroborate
azoospermia observed in the
1st sample.

PFOA, PFOSA, and MePFOSA-AcOH were mostoften associated with semen
parameters.
Adjusted for: age, parental
education, BMI, environmental
tobacco smoke exposure,
regular exercise, month ofsurveyTaipei, Taiwan

Semen was collected after ≥2days’ abstinence

Semen analysis was
performed one day aftercollection.

No significant associations with other semen
parameters

2 sperm chromatinstability measures
Zhou et al., 2016

Comments

β (95% CI) per ng/mL increase in PFNA:
Ln(T) –0.4233 (–0.6998, –0.1467) mmol/L
Ln(E2) 0.1252 (–0.0758, 0.3263) mmol/L

More associations between
individual PFAS levels andhormone levels were reported
in boys than in girls

From the control cohort of
the Genetics andBiomarkers study forChildhood Asthma

PFNA and its salts andPFDA and its salts

24

OEHHA
October 2021

Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Outcomes

Results

Serum reproductive
hormones and related
proteins: FSH, LH, T,
free T, E2, SHBG,
T/LH, free T/LH, T/E2,
FT/E2), inhibin B,
inhibin B/FSH
Semen quality: Spermconc., total sperm
count, semen volume,% motile sperm

Specific results for PFNA were not reported.
Authors state there were no associations between
semen quality and any PFASs.

Semen (after 2-day
abstinence)

Semen quality:

P-values were adjusted for false discovery
rate/multiple comparisonsMedian (IQR) 0.024 (0.013,
0.042)

sperm conc.,

Petersen et al., 2018

Serum

Faroe Islands

Median (range)

2007-20090.49 (0.61, 18.10)

Cross-sectional
N=263
Entire population of 24-26
year old men were invited

Pan et al., 2019
Nanjing, China
2015-2016
Cross-sectional
N=664 males from couples
who visited a reproductive
medical center. Some men
had fecundity issues andsome were partners of
women with female factor
infertility

Serum (same day as
semen):
median (IQR) 1.466 (1.011,
2.216)

semen volume,
sperm count,progressively motile(%), VCL,
VSL, morphologicallynormal (%), DNA
fragmentation index(DFI), HDS
Concentration andmotility were
measured by CASA

Exclusions: reproductive
tract disease, medication for
fertility, insufficient semen
volume for analysis

Associations with reproductive hormones were notmentioned for PFNA.

Covariates, other
PFASs analyzed
Reproductive hormones (except
FSH, LH) were adjusted for BMI,
smoking, age, time of day ofblood sampling.
Sperm conc., total sperm count,and semen volume were
adjusted for abstinence (hours).

Other PFASs: PFHxS, PFOA,PFOS, PFDA

Change (95% CI) per unit increase in:Ln(semen PFNA):
Progressively motile (%) β=–1.999 (–3.284, –0.714) p=0.02
VCL (µm/s): β=–0.792 (–1.542, –0.042) p=0.1
VSL (µm/s) β=–0.686 (–1.315, –0.058) p=0.1
DFI (%) β=0.106 (0.047, 0.165) p=0.01
HDS (%) β=0.044 (0.002, 0.086) p=0.1
No associations with other semen parameters
Ln(serum PFNA):

Adjusted for:Age, BMI, BMI2, smoking status,
alcohol use, abstinence time
Also considered: having
fathered a pregnancy,
occupational hazards, medical
history
Analyses of associations withsemen parameters focused on
the most abundant PFASs inserum and semen and those
detected in at least 80% ofsemen samples:
PFOA, PFOS, 6:2 Cl-PFESA,PFNA, PFDA, PFUnDA

No associations with other semen parameters.

Nanjing, ChinaMedian (IQR) 1.47 (1.03,
2.23)

2015-2016

PFNA and its salts andPFDA and its salts

Serum reproductive
hormones and related
proteins: total T, freeT, E2, FSH, LH, T/LH

% change (95% CI) in hormone level per unitchange in ln(serum PFNA), for all men and men <30 years, with p-trend for analyses by PFNA
quartiles:
Total T –3.99 (–7.01, –0.87), p-trend = 0.013

25

Correlation between serumand semen PFNA: r=0.72
Correlations between serum
PFNA and other PFASs:
PFHxS r=0.361
PFOA r=0.570
PFOS r=0.740
6:2 Cl-PFESA r=0.752
8:2 Cl-PFESA r=0.665
PFDA r=0.825
PFUnDA r=0.910
PFDoA r=0.821
PFTrDA r=0.810
Associations of PFNA withsemen parameters weresimilar to those of other
PFASs.

HDS (%) β=0.119 (0.063, 0.176) p=0.01

Serum

49% of those reached byphone (24% of all young men
invited by mail) participated.

% motile sperm was adjusted forinterval between ejaculation andassessment.

Morphologically normal (%) β=–0.262 (–0.545,
0.020) p=0.2

Cui et al., 2020

Comments

Adjusted for: age, BMI, smoking
status, time of blood sampling,
fasting status

No statistical adjustment for
multiple tests

OEHHA
October 2021

Reference; study
location, period,
design, sample
Cross-sectional
N=651 (see Pan et al., 2019)
Additional exclusion: missing
reproductive hormone data

Exposure matrix
and concentration(ng/mL)

Outcomes

Results

Mean (range) 1.82 (0.27,
17.30)as an indicator of
Leydig cell function

Semen sampled after 2day abstinence:

SHBG, FAI

< 30 years: –5.54 (–9.09, –1.84), ptrend=0.004
Free T –2.77 (–5.69, 0.25), p-trend = 0.072< 30 years: –4.54 (–8.90, 0.02), p-trend=0.016
E2 –2.08 (–7.56, 3.73), p-trend=0.475< 30 years: –5.32 (–11.40, 1.19), ptrend=0.107
SHBG –4.32 (–8.12, –0.37), p-trend = 0.033< 30 years: –4.50 (–8.00, –0.87), ptrend=0.051
Total T/LH ratio –3.4 (–7.92, 1.34), p-trend = 0.157< 30 years: –1.65 (–7.18, 4.21), p-trend=0.573
FAI, FSH, LH: no associations

Median (IQR) 0.02 (0.01,
0.04)
Mean (range) 0.03 (<LOQ,
0.36)

Ln(semen PFNA):
Total T –5.27 (–8.27, –2.18), p-trend = 0.001< 30 years: –7.18 (–10.97, –3.24), ptrend=0.001
Free T –2.76 (–5.71, 0.29), p-trend=0.075< 30 years: –8.45 (–12.96, 3.71), ptrend=0.062
E2 –2.97 (–8.45, 2.84), p=0.308< 30 years: –6.49 (–13.02, 0.54), ptrend=0.069
SHBG –6.44 (–10.17, –2.55), p-trend=0.001< 30 years: –3.82 (–7.67, 0.19), p-trend=0.001
Total T/LH ratio–6.19 (–10.61, –1.56), ptrend=0.009< 30 years: –6.32 (–12.02, –0.24), ptrend=0.042
FAI, FSH, LH: no associations

PFNA and its salts andPFDA and its salts

26

Covariates, other
PFASs analyzed

Comments

Also considered: abstinencetime, alcohol use, having
fathered a pregnancy

Spearman correlation
between serum and semen
PFNA r=0.716

Focused on the most abundant
PFASs (those that accounted for
70% and 73% % of PFASs inserum and semen samples,
respectively): PFOA, PFOS, 6:2
Cl-PFESA, PFNA

Associations were stronger inyounger men. No SS
associations among men >30
years.
P-values for associations withserum PFAS were greaterthan for semen PFAS.
Authors note that most
toxicological studies
demonstrate that PFAS
(including PFNA) exposure
reduces T secretion.
Effect sizes were greater but
less likely to reach statisticalsignificance in analyses
restricted to men who had
fasted.

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October 2021

Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Ma et al., 2021

PlasmaHangzhou, Zhejiang, ChinaMedian (IQR) [range]

2017

2.3 (1.7,3.9) [0.7, 16.7]

Prospective cohort IVF
outcomes) and crosssectional (hormones andsemen quality outcomes)

Concentrations by tertiles:

N=96 couples undergoing
IVF treatment due to tubal
factor infertility

1st: 0.7-1.8
2nd: 1.8-3.4
3rd: 3.4-16.7

Outcomes

Results

Plasma reproductive
hormones: FSH, LH,
E2, T

Compared to 1st tertile of PFNA:

Semen quality:
volume; sperm
motility, concentration,
motility, morphology
IVF outcomes

Hormones
No associations with FSH, LH, E2, or T
Semen quality
Sperm concentration ↓ in 2nd and 3rd (25%reduction) tertiles p-trend = 0.031

Covariates, other
PFASs analyzed
Adjusted for age, BMI, smoking
statusOther PFASs: PFBA, PFHxS,
PFHpA, PFOA, PFOS, PFOSA,PFNA, PFUnDA, PFDoA

Each couple contributed one
cycle of IVF treatment to thestudy.
Male and female partners’
PFNA levels were highly
correlated, r=0.74.
PFNA was strongly correlatedwith:

Sperm count ns ↓ in 2nd and 3rd tertiles, p-trend =0.050

PFDA r = 0.881*% sperm with normal morphology ns ↓ in 2nd and3rd tertiles, p-trend = 0.109

Men with severe male-factor
infertility were excluded

Comments

PFOS r = 0.736*
PFUnDA r = 0.870*

Progressive motility no association*p< 0.05

IVF outcomes

2nd and 3rd tertile PFUnDA
was associated with ↑ T

2nd tertile exposure was associated with more
fertilization, p-trend = 0.667
No associations with number of good quality
embryos at day 3, implantation, clinical pregnancy,
or live births
Hardell et al., 2014
Sweden, 2007-2011
Case-control
N=200 cases of newly
diagnosed prostate cancerfrom one hospital
N=186 population-based,
matched controls with nohistory of cancer

Whole blood drawn after
diagnosis but before
treatment with radiation or
chemotherapy.
Mean, median (range)

Prostate cancer
Medical records

OR = 1.2, 95% CI (0.8, 1.8) for exposure above
control median

Adjusted for age, BMI, year ofsample.

Authors state that using 75th percentile exposure
cutoff resulted in somewhat higher ORs, but did notshow results.

Matched on age and geographic
area.

Cases 0.679, 0.612(0.0500, 4.6)
Controls 0.631, 0.572(0.0850, 2.1)

PFNA and its salts andPFDA and its salts

Other PFASs: PFHxA,
PFHxS,PFOA, PFOS, PFDA,
PFUnDA, PFDoDA

Participation rates were 60%
for controls and 79% forcases.
Gleason score, prostatespecific antigen, and a
combination of both were notassociated with PFASs.
Serum was sampled aftercancer diagnosis.

27

OEHHA
October 2021

Reference; study
location, period,
design, sample

Exposure matrix
and concentration(ng/mL)

Ducatman et al., 2015

Serum

Mid-Ohio Valley

Mean ± SD 1.47 ± 1.63

2005 – 2006
Cross-sectional
N = 12,988 males over 20
years of age with measured
PSA.

Outcomes
Serum PSA
concentration
PSA ≥ 4.0 was
considered clinicallysignificant

Results
Ratio of adjusted geometric mean ln(PFNA)
concentrations for PSA ≥ 4.0 vs PSA < 4.0, by age
group:
20-49 years: 0.85 (0.69, 1.06)
50-69 years: 1.04 (0.95, 1.13)
No relationship with PSA (continuous) in either age
group

Sample from the C8 Study,
which initially included those
who lived, worked, or went
to school in one of the six
water districts contaminatedwith PFOA from a chemical
facility; an estimated 81% of
eligible residents
participated

PFNA and its salts andPFDA and its salts

28

Covariates, other
PFASs analyzed
Stratified by age 20–49 years vs.
50–69 years and adjusted forgeometric mean age within age
strata (35 or 60 years), smoking
status, alcohol intake, and BMI.
Other PFASs: PFHxS, PFOA,
PFOS

Comments
Sample size in analysis isapproximately half of original
sample. Other than exclusionof men ≥70 years, reasons forthe reduced sample size are
not stated.
Unclear whether stratification
and adjustment wereappropriate and adequate foraddressing confounding by
age

OEHHA
October 2021

4.2 PFNA: Animal studies of male reproductive toxicity
Overview
The male reproductive toxicity of PFNA has been investigated in four studies in rats (Feng etal. 2009; Feng et al. 2010; Hadrup et al. 2016; NTP 2019), five studies in mice (Das et al.
2015; Singh and Singh 2019a, 2019b, 2019c, 2019d) and one study in zebrafish (Zhang et al.
2016) (See Table 4.2.2). Experiments in which PFNA was administered as part of a mixture ofchemicals are not included in this review.
Three of the studies conducted in rats were in adult or pubertal male Sprague Dawley (SD)rats and one was in pubertal male Wistar rats. The study by NTP treated nine- to 11-week-old
SD rats at five dose levels (ranging from 0.625 to 10.0 mg/kg-day) by daily gavage for 28 days
(NTP 2019), 2019 #917}. All animals in the NTP study were subject to histopathologicalevaluation; however, because of treatment-related mortality in the 5.0 and 10.0 mg/kg-day
dose groups (mortality: 8/10 and 10/10, respectively) only animals from the control and three
lower dose groups (0.625, 1.25 and 2.5 mg/kg-day) were included in the full toxicity
assessment. In the other two studies in SD rats, eight-week-old male rats were treated atthree dose levels (1, 3, and 5 mg/kg-day) with PFNA by daily gavage for 14 days (Feng et al.
2009; Feng et al. 2010).
The study by (Hadrup et al. 2016) exposed seven-week-old male Wistar rats to PFNA at threedose levels (0.0125, 0.25, 5 mg/kg-day) by daily gavage for 14 days, and also included
additional treatment groups (not discussed further here) where PFNA was administered
together with a mixture of 14 other chemicals (Hadrup et al. 2016). Plasma levels of sex
steroid hormones and testicular expression of messenger ribonucleic acid (mRNA) of selected
genes involved in steroidogenesis were measured; however, differences between “PFNA only”
treatment groups and vehicle controls were not always subject to statistical testing and forseveral of the mRNAs assessed data were not shown.
Three of the studies in mice were in prepubertal male Parkes mice (25 days-old at the start of
exposure), with one treating the animals at two dose levels (0.2, 0.5 mg/kg-day) by daily
gavage for 90 days (Singh and Singh 2019a) and the other two treating the animals at ten-foldhigher doses (2, 5 mg/kg-day) by daily gavage for 14 days (Singh and Singh 2019b, 2019c).
Another two studies in mice treated pregnant females during gestation and investigated theeffects on male pups (Das et al. 2015; Singh and Singh 2019d). The study by Singh and Singh(2019d) treated pregnant female Parkes mice from gestation day (GD) 12 to parturition at two
dose levels (2, 5 mg/kg-day) and assessed male pups on postnatal days (PND) 1 and 3. The
study by Das et al. (2015) treated pregnant CD-1 mice from GD 1-17 at four dose levels (1, 3,
5, 10 mg/kg-day), and assessed male fetuses from half of the dams on GD 17 and maleoffspring from the remaining dams for up to 41 weeks of age. In this study, full litter resorptions
occurred in all of the pregnant dams in the 10 mg/kg-day group.

PFNA and its salts andPFDA and its salts

29

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October 2021

The effects of 180-day exposure of adult zebrafish to three concentration levels of PFNA (0.01,
0.1, 1 mg/L) on gonadosomatic index, measures of fertility, sex steroid hormones, expressionof vitellogenin protein and mRNA, and expression of mRNA of selected genes involved insteroidogenesis or associated with the HPG axis were evaluated (Zhang et al. 2016).
Relevant major findings from these studies are discussed below, grouped by the type of effect(reproductive organ weight and histopathology, sperm parameters, hormonal effects, fertility or
reproductive performance, and development of the male reproductive system).
Organ Weight and Histopathology
Histopathological changes in the testis have been observed in rats and mice treated by gavage
with PFNA.
Key findings from the NTP 28-day rat study are summarized in Table 4.2.1 below (NTP 2019).
The animals were approximately 9-11 weeks old at the beginning of dosing. Testis weight was
reduced at 1.25 and 2.5 mg/kg-day doses by approximately 7% and 20%, respectively.
Epididymal weight was reduced at 0.625, 1.25 and 2.5 mg/kg-day (7%, 13% and 35%,
respectively). Increased incidence of germ cell degeneration, spermatid retention (inhibited
spermiation), and interstitial cell atrophy were observed in the testes of rats exposed to PFNAat doses of 2.5 mg/kg-day and above. Treatment with Wyeth-14,643, a peroxisome
proliferator-activated receptor alpha (PPARα) agonist, at doses up to 25 mg/kg-day did not
result in any obvious testicular damage in the same study, even though liver effects wereapparent.

PFNA and its salts andPFDA and its salts

30

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October 2021

Table 4.2.1 PFNA or Wyeth-14,643: Relevant key findings in male rats treated by gavage for 28 days (NTP 2019)
Treatment

Dose (mg/kg-day)Wyeth-14,643

PFNA
Doses (mg/kgday)

0

0.625

1.25

2.5

5.0a

10.0a

25.0b

No. of rats per
group

10

10

10

10

10

10

10

Survival rate

10/10

10/10

10/10

10/10

2/10

0/10

10/10

Body weight (g)

344±6

332±6

286±5**

193±9**No data

No data308±5**

Relative liver
weight (g)34.14±0.342.12±0.58**

54.47±0.59**

64.37±1.86**

No data

No data

53.90±1.67**

Hepatocyte
cytoplasmic
alteration

0/10

10/10**

10/10**

10/10**

9/9**

10/10**

10/10**

Hepatocyte
hypertrophy

0/10

7/10**

10/10**

10/10**

9/9**

10/10**

10/10**

Relative thyroid
weight (g)0.07±0.00

0.08±0.00

0.07±0.00

0.09±0.01**

No data

No data

Not reported

TSH (ng/dL)

20.33±2.3113.70±1.27

10.97±1.22**

10.16±3.35**

No data

No data

13.07±1.01**

T3 (ng/dL)

78.21±4.54

58.54±2.1184.93±2.94111.79±10.16

No data

No data

Not reported

Free T4 (ng/dL)

216±0.15

0.55±0.02**

0.33±0.01**

0.30±0.00**

No data

No data1.31±0.13**

Total T4 (ng/dL)

2.36±0.0.27

0.21±0.07**

0.38±0.07**

1.49±0.13

No data

No data2.78±0.17**

Absolute testis
weight (g)1.885±0.041

1.820±0.035

1.762±0.031*

1.507±0.052**

No data

No data

1.774±0.036

Absolute
epididymal
weight (g)0.555±0.013

0.515±0.010*

0.482±0.005**

0.363±0.022**

No data

No data

0.512±0.011

Spermatid heads(106/testis)230.2±10.7

192.5±7.1*220.8±10.8205.1±9.5

No data

No data

255.8±11.5

PFNA and its salts andPFDA and its salts

31

OEHHA
October 2021

Treatment

Dose (mg/kg-day)Wyeth-14,643

PFNA
Doses (mg/kgday)

0

0.625

1.25

2.5

5.0a

10.0a

25.0b

Epididymal
sperm count(106/cauda
epididymis)

142.3±9.4

136.2±7.9116.0±6.3*

98.1±9.0**

No data

No data

116.3±8.4

Epididymal
sperm motility

85.2±0.585.9±0.4

86.4±0.75

86.4±0.7

No data

No data88.2±0.5

T (ng/mL)

4.48±1.31

4.86±1.33

3.23±1.387

0.85±0.51**

No data

No data1.56±0.39*

Interstitial cell
atrophy

0/10

0/10

1/10

10/10**

9/9**

10/10**

Not reported

Spermatid
retention

0/10

0/10

0/10

6/10**

9/9**

10/10**

Not reported

Germ celldegeneration

0/10

0/10

0/10

6/10**

9/9**

10/10**

1/10

Epididymal
lesion

0/10

0/10

0/10

6/10**

9/9**

10/10**

1/10

Due to high treatment-related mortality in the 5- and 10-mg/kg-day dosing groups, animals in these groups were subject to histopathology, butwere not assessed for other measures. b Only the findings from the high dose of Wyeth-14,643 are presented in this table. * p<0.05 **p<0.01

aPFNA and its salts andPFDA and its salts

32

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October 2021

Treatment with PFNA at 1 or 3 mg/kg-day for 14 days did not cause histopathological changes
in the testes of 8-week-old SD rats (Feng et al. 2009). At 5 mg/kg-day, PFNA caused
sloughing of germ cells into the lumen of the seminiferous tubules and chromatin condensationand margination in germ cells, characteristic of germ cell apoptosis (Feng et al 2009). In afollow-up study of the same design, ultrastructural changes were observed in the testes of rats
treated with PFNA at 3 mg/kg-day for 14-days, with an increased presence of vacuoles
between Sertoli cells and spermatogonia, and in the cytoplasm of the Sertoli cells.(Feng et al.
2010). Consistent with the earlier study, germ cell degeneration was observed in rats treatedwith PFNA at 5 mg/kg-day (Feng et al. 2010).
Prepubertal exposure to PFNA at 2 and 5 mg/kg-day in male Parkes mice from PND 25 to 38(14 days) caused histopathological damage to the testis (Singh and Singh 2019b, 2019c). The
lesions include increased vacuolation in the seminiferous epithelium, marginal chromatincondensation in round spermatids, multinucleated giant cells and sloughing of germ cells into
the lumen of seminiferous tubules. In comparison to the controls, the testis weights in the 2and 5 mg/kg-day dosing groups were reduced by approximately by 14% and 20%,
respectively, but the changes in testis weight were not statistically significant. In another study
in the same strain of mice by the same authors, degenerative germ cells, intraepithelial
vacuolation, and exfoliation of germ cells were observed in mice treated with 0.5 mg/kg-day of
PFNA for 90 days, with no effect on the testis weight (Singh and Singh 2019a). No effects on
the testis were observed at 0.2 mg/kg-day. In both studies, the number of germ cells withpositive staining for proliferating cell nuclear antigen (PCNA) was significantly reduced in thetestes of mice treated with PFNA at either 2 or 5 mg/kg-day for 14 days (Singh and Singh2019b) or 0.5 mg/kg-day for 90 days (Singh and Singh 2019a).
In zebrafish, exposure to PFNA at concentrations as low as 0.01 mg/L for 180 days caused
significant reduction in gonadosomatic index, indicative of reduced relative weight of malegonads in zebrafish (Zhang et al. 2016).
PFNA-induced germ cell degeneration by apoptosis was evaluated in the study by (Feng et al.
2009). The authors found increased number of TUNEL-positive germ cells, mainly inspermatocytes and spermatogonia, in the testes of rats treated with PFNA at 3 and 5 mg/kgday doses. Flow cytometry revealed an increase in apoptotic germ cells at 3 and 5 mg/kg-day.
Both mitochondria-dependent and caspase-8-dependent apoptotic pathways may be involved
in PFNA-induced germ cell apoptosis, as indicated by changes in the gene expression of Bcl2
associated X (Bax,up-regulated), B-cell lymphoma 2 (Bcl-2,(down-regulated), Fas (upregulated), and Fas Ligand (FasL,down-regulated), and increased protein levels of active
caspase-8. In mice treated with PFNA at 5 mg/kg-day for 14 days, flow cytometry of germ cell
populations found decreased relative population size of 4C germ cells, including leptotene,
zygotene, pachytene and diplotene primary spermatocytes and decreased spermatogonial
cells in G2 phase (Singh and Singh 2019c).

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Several studies discussed above found that PFNA at the dosing levels toxic to rat testes also
caused reduced food intake and body weight, liver damage, or other toxic effects. As cited in
the 2019 NTP study report, Chapin and Creasy had reported that decreased body weight gain
and/or decreased food intake may result in decreased T production through decreased GnRH
release and cautioned that such effects should not be mistaken for a direct effect of the test
material on androgen balance (Chapin and Creasy 2012). Studies investigating the effects ofsevere food restriction in rats for as long as 17 weeks that resulted in reductions in bodyweight of up to 30% did not observe effects on fertility, testicular weight, or severe
histopathological changes (Chapin et al. 1993; Rehm et al. 2008). Therefore, PFNA-induced
testicular effects are unlikely to be secondary to the general toxicity observed in the same
animals.
Sperm Parameters
Sperm parameters were evaluated in rats and mice exposed to PFNA. PFNA caused
significant reduction in epididymal sperm counts in rats at 1.25 and 2.5 mg/kg-day following
28-day exposure (decreases of 20% and 30%, respectively) (NTP 2019). In mice, exposure to
PFNA at 0.5 mg/kg-day for 90 days caused reduction in sperm count (8.5±0.4 million per testis
vs. 13.52±1.15 million per testis in controls) and motility (66.8±5.82% vs. 90.8±2.58% in
controls) (Singh and Singh 2019a). Sperm viability, measured by optical density (OD) of sperm
suspension samples following staining with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5diphenyltetrazolium
bromide (MTT), was also reduced in these animals by about 65% (0.22±0.02 OD vs. 0.62±0.04
in controls). At 0.2 mg/kg-day, sperm count, motility and viability were also reduced, but the
reductions were not statistically significant (11.63±1.18 million per testis, 85.4±4.52% and
0.48±0.09 OD, respectively).
Hormonal Effects
PFNA has been shown to cause reduction in serum or plasma levels of T in rats (Feng et al.
2009; NTP 2019) and mice (Singh and Singh 2019a, 2019b). In rats, reduced serum levels of
T were observed in SD rats treated for 28 days at 2.5 mg/kg-day (by about 81%) (NTP 2019)or at 5 mg/kg-day for 14 days (by about 85.4%) (Feng et al. 2009). However, PFNA at 1
mg/kg-day for 14 days caused an 87.5% increase in pubertal rats, with no effect on serum T
level at 3 mg/kg-day (Feng et al. 2009). There was no effect on FSH or LH levels in the studyby (Feng et al. 2009). The results on hormonal levels from the study in Wistar rats by (Hadrup
et al. 2016) are not clear. The authors stated that plasma levels of androstenedione andtestosterone were decreased at 5 mg/kg-day, but no statistically significant differences fromcontrol levels were noted in Figure 3 of the publication. No effect on plasma
dihydrotestosterone (DHT) was reported. The authors did not mention differences from control
in plasma LH, FSH or prolactin levels associated with PFNA treatment; however, LH may be
reduced and FSH and prolactin may be increased at 5 mg/kg-day. In addition to the effects on
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serum levels of T, PFNA treatment for 14 days caused reduced levels of inhibin B at 1, 3 and 5
ma/kg-day doses and increased levels of E2 and AMH (also called Müllerian inhibiting
substance or MIS) at 5 mg/kg-day (Feng et al. 2009; Feng et al. 2010).
In mice, treatment with PFNA for 14 days at 2 and 5 mg/kg-day or 90 days at 0.5 mg/kg-day
caused reduced serum levels of T when treatment began before puberty (PND 25) (Singh andSingh 2019a, 2019b). The reduction was about 72% (14-day at 2 mg/kg-day), 85% (14-day at
5 mg/kg-day) or 31% (90-day at 0.5 mg/kg-day). Intratesticular T levels were also decreased
by 20% and 38% following 14-day exposure at 2 and 5 mg/kg-day, respectively (Singh andSingh 2019b). Similarly, intratesticular levels of T in neonatal (PND 3) mice were reduced
following treatment in utero with PFNA at 5 mg/kg-day from GD 12 to parturition) (Singh andSingh 2019b, 2019d).
In zebrafish, increases in serum T were observed in adult male (and female) zebrafish
exposed to PFNA at concentrations as low as 0.01 mg/L for 180 days (Zhang et al. 2016).
This same study also observed increases in serum E2 in male (and female) zebrafish at PFNA
concentrations of 0.1 and 1.0 mg/L (Zhang et al. 2016) and increased liver levels ofvitellogenin (VtG) in males at these same concentrations.
There are several in vivo animal studies that investigated the potential gene targets and/or
molecular pathways underlying the hormonal effects of PFNA. Detailed discussions on thesefindings and those from a few in vitro studies are summarized in Section 4.3. Briefly, PFNA
treatment has been shown to cause the following changes in enzymes or genes associatedwith sexual hormone production and/or function in testicular tissues:
•
•
•
•

•

•

Androgen receptor (AR): reduced mRNA at 2 and 5 mg/kg-day in mice exposed for 14
days (Singh and Singh 2019c).
Follicle-stimulating hormone receptor (FSHR): reduced mRNA at 0.1 and 1.0 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
Luteinizing hormone receptor (LHR): reduced mRNA at 0.01, 0.1 and 1.0 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
Steroidogenic factor 1 (SF1): reduced protein at 2 and 5 mg/kg-day X 14-day in mice
exposed for 14 days (Singh and Singh 2019b); reduced protein at 5 mg/kg-day in mice
exposed in utero (Singh and Singh 2019d).
Steroidogenic acute regulatory protein (StAR); reduced mRNA at 0.5 mg/kg-day in mice
exposed for 90 days (Singh and Singh 2019a); reduced protein at 2 and 5 mg/kg-day inmice exposed for 14 days (Singh and Singh 2019b); reduced protein at 2 and 5 mg/kgday in mice exposed in utero (Singh and Singh 2019d); reduced mRNA at 5 mg/kg-day
in rats exposed for 14 days (Hadrup et al. 2016); increased mRNA at 0.1 and 1.0 mg/L
in zebrafish exposed for 180 days (Zhang et al. 2016).
Cytochrome P450 family 11 subfamily A (CYP11a): reduced mRNA at 0.5 mg/kg-day inmice exposed for 90 days (Singh and Singh 2019a); reduced protein at 2 and 5 mg/kgday in mice exposed for 14 days (Singh and Singh 2019b); reduced protein at 5 mg/kg-

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•

•

•

•

day in mice exposed in utero (Singh and Singh 2019d); reduced mRNA at 5 mg/kg-day
in rats exposed for 14 days (Hadrup et al. 2016); increased mRNA at 1.0 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
3β-Hydroxysteroid dehydrogenase (3β-HSD): reduced mRNA at 0.5 mg/kg-day in mice
exposed for 90 days (Singh and Singh 2019a); reduced protein at 2 and 5 mg/kg-day inmice exposed for 14 days (Singh and Singh 2019b); reduced protein at 5 mg/kg-day inmice exposed in utero (Singh and Singh 2019d); reduced mRNA at 0.01, 0.1 and 1.0
mg/L in zebrafish exposed for 180 days (Zhang et al. 2016).
Cytochrome P450 family 17 (CYP17): reduced mRNA at 5 mg/kg-day in rats exposedfor 14 days (Hadrup et al. 2016); no effect on mRNA at 0.01, 0.1 or 1.0 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
17β-Hydroxysteroid dehydrogenase (17β-HSD): reduced mRNA at 0.5 mg/kg-day inmice exposed for 90 days (Singh and Singh 2019a); reduced protein at 2 and 5 mg/kgday in mice exposed for 14 days (Singh and Singh 2019b); reduced protein at 5 mg/kgday in mice exposed in utero (Singh and Singh 2019d); reduced mRNA at 5 mg/kg-day
in rats exposed for 14 days (Hadrup et al. 2016); increased mRNA at 0.1 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
Cytochrome P450 family 19 subfamily A (cyp19a): increased mRNA at 0.1 mg/L inzebrafish exposed for 180 days (Zhang et al. 2016).
Fertility or Reproductive Performance

Reductions in fertility index and litter size were observed when unexposed female mice weremated with males exposed to 0.5 mg/kg-day PFNA from PND 25 to 114 (90-day exposure), but
the design details for the fertility test in this study were not reported (Singh and Singh 2019a).
Exposure of pregnant mice to PFNA at doses of 2 or 5 mg/kg-day from GD 12 until parturition
did not cause significant changes in birth rate, number of pups per dam, sex ratio of pups or
the birth weight of male pups in mice, though obvious adverse effects were found in neonatal
mice following gestational exposure (e.g., survival, body weight gain) (Singh and Singh
2019d).
The weekly fertility assessments conducted throughout the 180-day PFNA exposure period in
the adult zebrafish study by Zhang et al. (2016) found that the number of eggs per matedfemale was significantly reduced at 0.1 mg/L (approximately 210 eggs per female vs. 250 incontrols), but not at 0.01 or 1.0 mg/L. In addition, the hatching rate of eggs at the 72-hr time
point was reduced at 0.01 mg/L and 1.0 mg/L (approximately 70% vs. 80% in controls), but not
at 0.1 mg/L. Since both male and female fish were exposed to PFNA in this study, thereduction in egg production and hatching rate could have resulted from effects in either female
or male animals, or in both.

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Development of the Male Reproductive System
Developmental exposure to PFNA has been shown to cause adverse effects in the testes ofneonatal mice. In the study by (Singh and Singh 2019d), gestational treatment with PFNA at 2
and 5 mg/kg-day reduced testes weights (1.05 ± 0.09 and 0.99 ± 0.13 mg, respectively), incomparison to 1.31 ± 0.11 mg in the controls. The reduction, approximately 20% and 24%,
respectively, was not statistically significant, but there were only 10 pups from five litters pergroup, a relatively small sample size for this parameter. Intratesticular T levels in the twotreated groups were also reduced in the 5 mg/kg-day dose group (1.07 ± 0.07 ng/g tissue
vs.1.83 ± 0.105 ng/g tissue in the control), but not in the 2-mg/kg-day group (1.70 ± 0.10 ng/g
tissue, 3 pooled tissue samples per group). The authors did not find histopathological changes.
As shown in Figure 1 of the paper, seminiferous cords of the mouse testes on PND 3 consist
mainly of proliferating Sertoli cells with very few large gonocytes in quiescence (Auharek andde França 2010). Therefore, there should be no apparent germ cell degeneration in neonatal
testis in mice, assuming the targeting cells in the rodent testis are 4C spermatocytes (Singhand Singh 2019c). In addition, the authors found reduced protein level of PCNA in testicular
tissues from neonatal mice exposed gestationally to 5 mg/kg-day, indicative of inhibited
proliferation of Sertoli cells. Decreased protein expression of steroidogenic genes (e.g. StAR,
CYP11A1, 3β‐HSD, 17β‐HSD and SF1) as well as alteration in the expression of WT1, SF1
and Insl‐3 were also observed in the testes from mice exposed to 5 mg/kg-day gestationally.
In addition to the study by Singh and Singh (2019d), preputial separation was significantly
delayed in male mice following gestational exposure of the dams (GD 1- 17) to PFNA at 3.0
and 5.0 mg/kg-day (Das et al. 2015; Singh and Singh 2019d).

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Table 4.2.2 PFNA: Evidence on the male reproductive toxicity in animal studies.
Study Design
NTP 2019
Male SD rats, 9-11 weeks old at the beginning of
dosing, 10 rats per dose group.
Treatment: PFNA (99.5% purity) once daily by gavage
in deionized water with 20% Tween® 80 at doses of0, 0.625, 1.25, 2.5, 5.0 or 10.0 mg/kg-day,respectively, for 28 days. Six other PFASs and Wyeth14,643 (a PPARα agonist) were alsotested forcomparison.

Outcomes Assessed

Major Findings

Notes

General toxicity: survival, body weights,
clinical chemistry, thyroid hormones, liver
effects (e.g., gene expression, enzyme
activity) and histopathology.

General toxicity: Decreased survival in the 5 and 10 mg/kg-day
dose groups (2/10 and 0/10, respectively). All animals in thecontrol and other dose groups survived through the 28-day
treatment period. Lower body weights at ≥ 1.25 mg/kg-day.
Increased relative weights of liver, kidney, and adrenal gland;
increased hepatic expression of PPARα -associated genes(Acox1, Cyp4a1) and constitutive androstane receptor (CAR)associated genes (Cyp2b1, Cyp2b2) at ≥ 0.625 mg/kg-day.

Treatment period of 28 days isshorter than one full cycle ofspermatogenesis in rats (about 56
days).

Male reproductive toxicity: testis andepididymis weights; sperm production
and motility; histopathology of the testis
and epididymis; serum T.

Male reproductive toxicity:
At 0.625 mg/kg-day: reduction in epididymis weight (7%) andcount of spermatid heads per testis (14%); no histopathologicallesions in the testis or epididymis.

Similar effects were observed withPFDA treatment.
Treatment with Wyeth-14,643 caused
significant effects in the liver, but notin the testis, indicating testiculareffects of PFNA may not be mediated
via the PPARα pathway.

At 1.25 mg/kg-day: reduction in testis weight (7%), epididymis
weight (13%), and cauda epididymal sperm counts. Minor
interstitial cell atrophy in one of 10 rats.
At 2.5 mg/kg-day: serum T reduced by 81%; reduction in testis
weight (20%), epididymis weight (35%), and cauda epididymal
sperm counts (30%).
Of 10 rats in the group, 6 had germ cell degeneration; 6 had
exfoliated germ cells in epididymal ducts, and all 10 had
interstitial cell atrophy.
At 5 and 10 mg/kg-day: all rats had histopathological lesions in
the testis or epididymis.

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Study Design

Outcomes Assessed

Major Findings

Feng et al. 2009

Male reproductive toxicity:

Male reproductive toxicity:

Male SD rats, aged 8 weeks at the beginning oftreatment; six animals per group.

Serum levels of T, E2, FSH and LH.

Serum T level significantly increased (by 87.5%) at 1 mg/kg-day,significantly reduced at 5 mg/kg-day (by 85.4%). No effect at 3
mg/kg-day.

Treatment: PFNA (97% purity) in water (containing
0.2% Tween-20), daily gavage for two weeks at doses
of 0, 1, 3, or 5 mg/kg-day, respectively.

Testis histopathology; gene expression of
Bax, Bcl-2, Fas and FasL; western
blotting analysis on caspase-8 andcaspase-9 proteins.

No effect on serum levels of FSH or LH at any dose.
Increased E2 at 5 mg/kg-day.

Notes
No data on general toxicity were
included in the study report.
The animals were slightly younger at
the start of exposure (8 vs. 9-11
weeks of age), and the treatment
period was shorter (2 vs 4 weeks), incomparison to the NTP study.

Germ cell degeration and sloughing of seminiferous epithelium at
5 mg/kg-day, but not at 1 or 3 mg/kg-day.
Germ cell flow cytometry revealed increased germ cell apoptosis
at 3 and 5 mg/kg-day.
Effects observed on the testicular expression of genes andproteins involved in apoptosis pathways:
Increase in Fas and Bax mRNA at 5 mg/kg-day; decrease in FasL
at 3 mg/kg-day; decrease in Bcl-2 mRNA at 3 and 5 mg/kg-day;dose dependent increase in active caspase-8 (statisticallysignificant at 3 and 5 mg’kg-day). No effect on active caspase-9.
Feng et al. 2010

Male reproductive toxicity:

Male reproductive toxicity:

Male SD rats, 8 weeks old, 6 rats per group.

Serum levels of Müllerian inhibiting
substance (MIS) and inhibin B.

Serum MIS was significantly increased at 5 mg/kg-day.

Treatment: PFNA (97% purity) in water (containing
0.2% Tween-20), daily gavage for two weeks at doses
of 0, 1, 3, or 5 mg/kg-day, respectively.

Testicular ultrastructure by electron
microscopy.
Analysis of protein expression by western
blotting: Wilms’ tumor gene (WT1) andtransferrin.

Serum inhibin B was significantly reduced at all doses.
Treatment with 3 or 5 mg/kg-day caused increased presence of
vacuoles of different sizes either between Seroli cells andspermatogonia or in the cytoplasm of Sertoli cells, indicative of
disruption of tight junctions between Sertoli cells and/or
degenerative changes in the seminiferous epithelium. No datareported on the ultrastructure of the testes from animals treatedwith 1.0 mg/kg-day.

No data on general toxicity were
included in the study report.
Study design for the in vivo animal
experiment is similar to that used in
the Feng et al. 2009 study. The paper
also reports results from in vitro
experiments conducted with primary
Sertoli cells. Findings from the in vitro
experiments are summarized in
Section 4.3.

Germ cell degeneration shown at 5 mg/kg-day
Increased WT1 protein and reduced transferrin protein in testicular
tissues at all doses.

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Study Design
Hadrup et al. 2016
Male Wistar rats, 7 weeks old, 8-10 rats per group.
Treatment: PFNA (purity not stated) in corn oil at
doses of 0, 0.0125, 0.25 or 5.0 mg/kg-day by gavagefor 14 days. Additional groups of rats were treatedwith a mixture of 14 chemicals in the presence orabsence of PFNA. Findings from exposures to thesehecmical mixtures are not discussed further here.

Outcomes Assessed

Major Findings

Notes

General toxicity: body weight, liver
histology (cell size and cell borderclearness), plasma levels ofcorticosterone, ACTH, and brain-deprived
neurotrophic factor, mRNA levels ofsteroidogenesis enzymes in liver andadipose tissues.

General toxicity: Body weight was reduced, and liver cell borders
were less apparent at 5.0 mg/kg-day. Nonstatistically significant
increase in liver cell size (hypertrophy) with increasing dose.
Increased plasma corticosterone at 0.0125 mg/kg-day, but not at
higher doses. No effect on plasma levels of ACTH or braindeprived neurotrophic factor. The authors reported changes inmRNA levels of genes involved in steroidogenesis in liver andadipose tissue at 5 mg/kg-day, but the data were not presented.

Differences in between “PFNA only”
treatment groups and vehicle controls
were not subject to statistical testingin many instances, e.g., for plasma
LH levels, for tissue-specific mRNAlevels.

Male reproductive toxicity: plasma levelsof androstenedione, T, DHT, LH, FSH
and prolactin.
mRNA levels of 17β-HSD, StAR,
Benzodiazepine receptor (BZRP; i.e.,
mitochondrial translocator protein or
TSPO), CYP11A, and CYP17 in testicular
tissues.

Male reproductive toxicity: The authors stated that plasma levels of
androstenedione and testosterone were decreased at 5 mg/kg-day,
but no statistically significant differences from control levels werenoted in Figure 3. No effect on plasma DHT. The authors did not
mention differences from control in plasma LH, FSH or prolactin
levels associated with PFNA treatment; however, Figure 4
suggests that LH may be reduced and FSH and prolactin may be
increased at 5 mg/kg-day.
No effect of 0.0125 or 0.25 mg/kg-day on testicular mRNA levels of
17β-HSD. At 5 mg/kg-day, testicular mRNA levels of 17β-HSD,
StAR, BZRP (TSPO), CYP11A, and CYP17 were decreased (datawere not shown).

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Study Design

Outcomes Assessed

Major Findings

Singh and Singh 2019a

General toxicity: body weight

General toxicity: No effect on body weight.

Male Parkes mice, 25 days old. 14 animals per group.
Seven males per group were randomly selected for
fertility test at the end of treatment. One male wasmated to one prooestrous female overnight and themated females were allowed to complete the term forlitters. The remaining seven males were sacrificed fortesticular weights and tissue samples. Only five fromeach group were included for sperm evaluation.

Male reproductive toxicity: Epididymal
sperm analysis: number, motility, andviability of sperms from cauda
epididymidis of five animals per group.

Male reproductive toxicity: No effect on testicular weight at any
dose.

Treatment: PFNA (97% purity) in distilled water, daily
gavage at doses of 0, 0.2, or 0.5 mg/kg-day,respectively, from PND 25 to 114 (for 90 days). The
animals were sacrificed on PND 115.

Testis histopathology.
Serum T levels.
Analysis of mRNA expression bysemiquantitative RT-PCR: StAR,
CYP11A1, 3β‐HSD, 17β‐ HSD in
testicular tissues.

Five mice per group were selected forsperm evaluation. Group size isrelatively small for these parameters.

At 0.2 mg/kg-day:Reduced SOD and catalase activity in testes. Sperm number,
motility and viability were reduced (17%, 6% & 23%, respectively),
but did not reach statistical significance. No effect on otherparameters evaluated (serum T level; expression of genes orproteins). No histopathological changes in the testes. No effect onmating index, fertility index or litter size.
At 0.5 mg/kg-day:

Immunoblotting: StAR, CYP11A1, 3β‐
HSD, 17β‐ HSD, PCNA & caspase‐3 intesticular tissues.

Degenerative changes in the seminiferous epithelium: loosening ofgerminal epithelium, intraepithelial vacuolation, decreased heightof germinal epithelium, and exfoliation of germ cells.

Immunohistochemical staining: PCNA in
testicular tissues.

Decreased PCNA positive gern cells. Reduced sperm number,
motility & viability (by approximately 37%, 26% and 65%,
respectively).

Activities of superoxide dismutase (SOD),
catalase & glutathione-s-transferase(GST) in testicular tissues.

Notes

Reduced serum T level (31%).
Fertility index was reduced (5/7 mice or 71.42% vs. 7/7 mice or
100.00% in the controls).
Significantly reduced litter size (4.6±1.12 vs. 8.0±1.00 in controls,
p<0.05).
Testes: Down-regulation in mRNA levels of StAR, CYP11A1, 3β‐
HSD and 17β‐ HSD. Decreased protein levels of StAR, CYP11A1,
3β‐HSD, 17β‐ HSD and PCNA. Increased protein levels of
caspase-3. Increased lipid peroxidation and decreased SOD,
catalase and GST activity.

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Study Design

Outcomes Assessed

Major Findings

Singh and Singh 2019b

General toxicity: Body weight.

General toxicity: Reduced body weight gain (24%) at 5 mg/kg-day.

Male Parkes mice, 25 days old (prepubertal). 10
animals per group. Five animals from each groupwere randomly selected for histology andhistochemistry, and remaining five for biochemistry,
immunoblotting and measurement of intratesticular
testosterone levels.

Male reproductive toxicity: Testis weight.

Male reproductive toxicity: Absolute testis weights reduced by 14%
and 20% at 2 and 5 mg/kg-day, respectively, but not statisticallysignificant.

Treatment: PFNA (97% purity) in distilled water, daily
gavage at doses of 0, 2, or 5 mg/kg-day for 14 days
(PND 25 to 38). The animals were sacrificed on PND
39.

Testis histopathology.
Germ cell apoptosis by TUNEL.
Germ cell proliferation by PCNA
immunostaining
Serum and intra-testicular levels of T.
Testicular protein levels by
immunoblotting: SF1, StAR, CYP11A1,
3β-HSD, 17β- HSD, nuclear factorerythroid-2-related factor-2 (Nrf2), heme
oxygenase-1 (HO-1), nuclear factor
kappa B (NF-κB), inhibitor of NF-κB
kinase (IKKβ), caspase-3, PCNA, and αtubulin
Testicular lipid peroxidation and SOD,
catalase and GST activity.

PFNA and its salts andPFDA and its salts

Notes

Testicular histopathological lesions at both doses: intraepithelial
vacuolation, marginal condensation of chromatin in roundspermatids, multinucleated giant cells and exfoliation of germ cells.
Increased number of apoptotic germ cells and decreased PCNA
positive germ cells at both doses.
Serum T decreased by 72% and 85%, at 2 and 5 mg/kg-day,respectively. Intra-testicular T decreased by 20% and 38%, at 2
and 5 mg/kg-day, respectively.
Significant changes in protein levels in testicular tissues at bothdoses: Decreased SF1, StAR, CYP11A1, 3β-HSD, 17β-HSD, Nrf2,
HO-1, PCNA; increased IKKβ, NF-κB, caspase-3.
Increased testicular lipid peroxidation and decreased SOD,
catalase and GST activity.

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Study Design
Singh and Singh 2019cMale Parkes mice, 25 days old. 10 animals per group,
Treatment: PFNA (97% purity) in distilled water, daily
gavage at doses of 0, 2, or 5 mg/kg-day for 14 days
(PND 25 to 38).

Outcomes Assessed

Major Findings

General toxicity: Liver weight, histology,
lipid peroxidation, PPARα mRNA. Serumcholesterol, blood glucose

General toxicity: Increased absolute and relative liver weight andhypertrophy at both doses; increased liver lipid peroxidation and
PPARα mRNA levels at both doses.

Male reproductive toxicity: Germ cell
populations by flow cytometry. Germ cells
were classified as 1C-round spermatids,
2C spermatogonial cells in G1 phase, Sphase-preleptotene spermatocytes andproliferative spermatogonia, and 4Cleptotene, zygotene, pachytene anddiplotene primary spermatocytes andspermatogonial cells in G2 phase.

Decreased cholesterol at 5 mg/kg-day.

Testes mRNA levels (qRT-PCR):
peroxisome proliferator-activated receptoralpha (PPAR α), scavenger receptor
class B type I (SR-B1), 3-hydroxy-3methylglutaryl coenzyme A synthase 1(HMG CoA synthase 1), growth hormone
receptor (GHR), insulin like growth factor
1 (IGF-1), insulin like growth factor 1
receptor (IGF-1R), AR.

No effect on relative population size of 1C or S-phase cells.

Testicular histology: staining for lactate
dehydrogenase type c (LHDC)
Testicular levels of lactate
dehydrogenase (LDH) activity, glucose,and lactate.

Notes

Increased blood glucose at both doses.
Male reproductive toxicity:
Germ cell populations:decreased relative population size of 4C cells at 5 mg/kg-day;
Increased relative population size of 2C cells at both doses.
Decreased 1C:2C at both doses.
Increased 1C:4C ratio at 5 mg/kg-day.
Reduced mRNA levels of HMG CoA synthase 1, SR-B1, GHR,
IGF-1R, AR and PPARα at both doses, and of IGF-1 at 5 mg/kgday.
Decreased testicular staining for LDHC (expressed primarily inspermatocytes and spermatids) at both doses.
Decreased testicular LDH activity, and glucose and lactate levels
at both doses.
Decreased testicular protein levels of LDHC, GLUT-3 and p-mTOR
at both doses.

Testicular protein levels of LDHC,
glycose transporter 3 (GLUT-3), andphosphorylated mammalian target of
rapamycin (p-mTOR)

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Study Design
Singh and Singh 2019dPregnant female Parkes mice, 10 per group. For testis
evaluation, 2 male pups from each dam (a total of 20
pups per group) were sacrificed on PND 3.
Treatment: PFNA (97% purity) in distilled water, daily
gavage to pregnant mice at 0, 2, or 5 mg/kg-day from
GD 12 until parturition.
One testis from each of five male pups per group wasprocessed for histology and immunohistochemistry.
The remaining testes from these 5 pups and all other15 male pups were used for immunobotting and othermeasurements.

Outcomes Assessed

Major Findings

General toxicity: dam body weight, birthrate, litter size, sex ratio of offspring, andbody weight of male pups on PND 1.

General toxicity: No effect on dam body weight or pregnancy
outcomes (birth rate, litter size, sex ratio of pups, or birth weight ofmale pups).

Male reproductive toxicity:

Male reproductive toxicity:
No effect on weight or histology of neonatal testis.

Testicular histology and staining for 3β‐
HSD.
Intratesticular T levels.
Testicular immunoblotting: StAR,
CYP11A1, 3β‐HSD, 17β‐HSD, WT1,
SF1, and PCNA.
Quantitative RT-PCR: mRNA expressionof c‐Kit, Insl‐3 and Amh, also called Mis).

Significant reduction in intratesticular levels of testosterone at 5
mg/kg-day (1.07 ± 0.07 ng/g tissue vs 1.83 ± 0.105 ng/g tissue incontrols, p<0.05)

Notes
Gonocytes are largely inactive on
PND 3, and thus germ celldegeneration, as observed by thesame authors in prepubertal mice,
may not be observed in newborn
mice. Majority of the cells within
seminiferous cords, as shown in
Figure 1A-1C, are Sertoli cells.

Decrease in protein level of StAR and WT1 at both doses, andCYP11A1, 3β‐HSD, 17β‐HSD, SF1 and PCNA at 5 mg/kg-day. A
reduction in 3β‐HSD protein at 5 mg/kg-day was also found byimmunohistochemical staining.
Decreased Insl-3 mRNA levels at both doses.
Decreased c-Kit and Amh (Mis) mRNA levels at 2 mg/kg-day;increased c-Kit and Amh (Mis) mRNA levels at 5 mg/kg-day.

Das et al. 2015
Pregnant female CD-1 mice and male offspring.
Treatment: PFNA (97% purity) in distilled water, daily
gavage to pregnant mice at 0, 1, 3, 5, or 10 mg/kg-day
from GD 1 to 17. No successful pregnancies occurredin the 10 mg/kg-day dose group. On GD 17 half thedams were sacrified for examination of fetuses, andhalf the dams were allowed to give deliver. For
postnatal growth assessment 4 male pups per litter
were examined, with 6-13 litters per dose group.

Toxicity in dams: body weight gain, liver
weight.
Developmental toxicity: pregnancy
outcome, fetal weight, fetal liver weight,
postnatal growth.
(Developmental) male reproductive
toxicity: preputial separation in malepups.

Toxicity in dams: Reduced maternal body weight gain at 10 mg/kgday. Increased maternal body weight gain at 3 and5 mg/kg-day,from GD 11 onwards. Increased absolute and relative liver weights
at 1, 3 and 5 mg/kg-day.
Developmental toxicity: Full litter resorptions in all pregnant dams
at 10 mg/kg-day (7/7). No effect on pregnancy outcomes at lower
doses. Increased fetal relative liver weight at 1, 3 and 5 mg/kg-day.

This study assessed multiple
measures of developmental toxicity,
however, only measures relevant tomale reproductive toxicity are
discussed in this document.

Reduced postnatal survival in male pups at 5 mg/kg-day. Reduced
body weight gain in male pups at 3 and 5 mg/kg-day that persisted
through at least 41 weeks of age. Increased relative liver weight inmale pups at all doses through PND 24, and at 3 and 5 mg/kg-day
through PND 42. Delayed eye opening in both sexes, and delayed
vaginal opening in female pups (by 3 and 7 days at 3 and 5 mg/kg-day, respectively, in comparison with controls).
(Developmental) male reproductive toxicity:
Preputial separation was delayed by 2 and 5 days in male pups at
3.0 and 5.0 mg/kg-day, respectively, compared to controls (meancontrol preputial separation on PND 28).

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Study Design
Zhang et al. 2016
Zebrafish, wild-type, Tuebingen strain, 5 months old.
A total of 480 fish of both sexes were included inthese studies. Gonadosomatic index (GSI) assessedin 30 fish/sex/dose. Six pairs of male and female fishfrom each dose group were randomly selected for
fertility assessment (egg production, fertilization rate,
hatching rate, and abnormality rate) every week.
Serum hormone measurements were performed on 3
pooled samples (pooled from 20 males) per dosegroup. mRNA analyses were performed on 6 males
per dose group.
Treatment: PFNA (97% purity) in water through aflow-through exposure system at concentrations of 0,0.01, 0.1 and 1.0 mg/L for 180 days.

Outcomes Assessed
Male reproductive toxicity: PFNA
concentrations in gonads.
GSI: gonad weight x 100/body weight).
Egg production and hatching rate of F1
generation
Serum levels of T and E2.
Vitellogenin mRNA (vtg) and protein (Vtg)
levels in male liver.
Transcriptional profiles of genes
associated with the synthesis of sex
hormones and the hypothalamic-pituitarygonadal-liver axis (HPGL axis):
Brain: estrogen receptor α (era), estrogen
receptor β (erb), ar, cytochrome P450
family 19 subfamily B (cyp19b),
luteinizing hormone β (lhb).
Gonad: fshr, lhr, star,
cyp11a, 3β-hsd, cyp17, 17β-hsd,
cyp19a.
Liver: era and erb.

PFNA and its salts andPFDA and its salts

Major Findings

Notes

Male reproductive toxicity:
PFNA concentrations in male spermary were approximately twice
as high as in female ovary.

This study evaluated adverse effects
of PFNA in zebrafish after chronic
exposure and focused on the
estrogenic effects of PFNA.

Reduced GSI in males at all dosing levels; no effect on GSI in
females.
Reduced egg production (by approximately 15%) at 0.1 mg/L.
Reduced hatching rate at 72-hr time point (by approximately 12%)
at 0.01 and 1.0 mg/L.
Increased serum T at 0.01 mg/L in males (and females). Increased
serum E2 at 0.1 and 1.0 mg/L in males (and females). The T/E2
ratio in males from the 0.01 and 0.1-mg/L groups appeared to be
increased (approximately 180 at 0.01 mg/L and 110 at 0.1 mg/L vs.
45 in controls), but statistical significance was not indicated.

Male zebrafish can affect female egg
production and hatching rate. Sinceboth males and females were
exposed to PFNA, the findings ofreduced egg production and hatchingrate could result from effects inmales, or females, or both.

Increased vtg mRNA levels in male liver at all dosing levels.
Increased Vtg protein levels in male liver at 0.1 and 1.0 mg/L.
Altered mRNA levels of genes related to the HPGL axis in males:Brainera: decreased at 0.01 mg/L, increased at 0.1 mg/Lerb:
decreased at 0.01 mg/L, increased at 0.1 mg/L
ar: decreased at 0.1 and 1.0 mg/L
cyp19b: no change
fshb: no change
lhb: increased at 0.1 mg/L
Gonad
fshr: decreased at 0.1 and 1.0 mg/L
lhr: decreased at all doses
star: increased at 0.1 and 1.0 mg/L
cyp11a: increased at 1.0 mg/L
3β-hsd: decreased at all doses
cyp17: no change
17β-hsd: increased at 0.1 mg/L
cyp19a: increased at 0.1 mg/L
Liverera: increased at 0.1 and 1.0 mg/L
erb: increased at 0.1 mg/L

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4.3 PFNA: Mechanistic considerations and other relevant data
The mechanistic literature regarding PFNA and male reproductive toxicity is reviewed here.
First, we will provide a brief overview of general toxicity findings from studies of PFNA,
followed by discussion of studies that may be more informative with respect to the effects
PFNA may have on the male reproductive system.
General toxicity
In male Sprague-Dawley rats administered PFNA at 0, 0.625, 1.25, 2.5, 5, or 10 mg/kg (oncedaily by oral gavage, 7 days a week) for 28 days, decreased body weight was observed in all
but the lowest dosed group, and survival was significantly reduced in the two highest dosegroups (NTP 2019). Other studies have observed similar effects of PFNA on body weight. A
study of male Wistar rats administered PFNA orally for 14 days reported a reduction in bodyweight at 5 mg/kg-d (Hadrup et al. 2016). In prepubertal male Parkes mice administered
PFNA for 14 days, a significant reduction in body weight gain was observed at 5 mg/kg-day(Singh and Singh 2019b); however, no effect on body weight gain was observed in male
Parkes mice exposed to lower doses of PFNA (0.2, 0.5 or 2 mg/kg-d) for 90 days (Singh andSingh 2019a). Reduced body weight gain was also observed in CD-1 mice exposed in utero to
PFNA (maternal doses of 3 and 5 mg/kg-day from GD 1-17), that persisted through at least 41
weeks of age (Das et al. 2015).
In vitro study in testicular cells
In a culture of testicular cells from seminiferous tubules of Wistar rats, exposure to PFNA (300µM for 48 hours) caused a significant increase in DNA damage (DNA strand breaks, asmeasured in the comet assay), in the absence of major effects on cell viability (Lindeman et al.
2012).
Effects on the hypothalamic-pituitary-gonadal-(liver) axis
The liver can be considered a component of the HPG axis in fish, as liver production ofvitellogenin, a precursor egg yolk protein, in male fish is under estrogenic control (Jo et al.
2014). Studies investigating the effects of PFNA on hormone levels, metabolism and receptormediated effects related to the hypothalalamic-pituitary-gonadal-liver (HPGL) axis are
summarized in Table 4.3.1.
Effects of PFNA on hormone levels have been studied in vivo in rats, mice and zebrafish andin vitro in cultured primary rat Sertoli cells. PFNA increased serum T levels in one study inmale SD rats exposed for two weeks at 1 mg/kg-day (Feng et al. 2009), and reduced serum T
levels in two studies in male SD rats exposed to higher doses (i.e., 2.5 mg/kg-day for 28 days
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(NTP 2019); 5 mg/kg-day for 2 weeks (Feng et al. 2009). PFNA also reduced serum T levels
in two studies in prepubertal male Parkes mice exposed for either 14 days at 2 and 5 mg/kgday (Singh and Singh 2019b) or 90 days at 0.5 mg/kg-day (Singh and Singh 2019a).
Intratesticular T levels were reduced in prepubertal male Parkes mice exposed to PFNA at 2
and 5 mg/kg-day for 14 days (Singh and Singh 2019b) and in PND 3 male Parkes mice
exposed to PFNA in utero (Singh and Singh 2019d).
In male SD rats PFNA at 5 mg/kg-day for 14 days increased serum levels of E2 (Feng et al.
2009) and MIS, also known as anti-mullerian hormone or Amh, involved in sex differentiation)
following treatment at 5 mg/kg-day for 14 days (Feng et al. 2010). Exposure to PFNA in utero
altered testicular gene expression of MIS in male Parkes mice on PND 3, with a decrease inmRNA observed in male pups of dams receiving 2 mg/kg-day (daily from GD 12 to parturition),and an increase in mRNA observed in male pups of dams receiving 5 mg/kg-day (Singh andSingh 2019d). Inhibin B, a glycoprotein produced by Sertoli cells that modulates FSH secretion(via a negative feedback loop), was decreased in male SD rats following treatment at 1, 3, and5 mg/kg-day for 14 days (Feng et al. 2010).
Data presented in Hadrup et al. (2016) suggest that PFNA may have reduced serum levels of
LH and increased serum levels of FSH and prolactin following 14-day exposure of male Wistar
rats at 5 mg/kg-day. No effects of PFNA on serum levels of LH or FSH were observed in a 14day study of male SD rats (Feng et al. 2009).
In male zebrafish, PFNA increased serum T at 0.01 mg/L, increased serum E2 at 0.1 and 1.0
mg/L, and increased the serum T to E2 ratio at 0.01 and 0.1 mg/L. PFNA also increased brain
mRNA levels of lutenizing hormone beta polypeptide (lhb), the gene encoding LH, at 0.1 mg/L
in male zebrafish (Zhang et al. 2016).
In cultured primary Sertoli cells isolated from 8 week old SD rats, in vitro exposure to PFNA for
24 hours increased MIS mRNA levels at 10, 25, 50, and 75 µM, and decreased inhibin B
mRNA levels at 50 and 75 µM (Feng et al. 2010).
Effects of PFNA on hormone synthesis and metabolism have been studied in vivo in rats,
mice, and zebrafish and in vitro in a human placental choriocarcinoma cell line and a mouse
Leydig tumor cell line.
Exposure of male rats to 5 mg/kg-day PFNA for 14 days was reported to reduce testicularmRNA levels of several steroidogenic enzymes (StAR, TSPO, CYP11A, CYP17, and 17βHSD), although the data were not shown, and it is unclear whether statistical analyses were
performed comparing “PFNA only” treatment groups and vehicle controls (Hadrup et al. 2016).
In a series of studies in male Parkes mice, PFNA was found to reduce testicular gene andprotein expression of a number of enzymes and factors involved in steroidogenesis.

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•

•

•

•

PFNA decreased mRNA levels of HMG Co synthase 1, an enzyme involved incholesterol synthesis, in prepubertal males exposed for 14 days to 5 mg/kg-day (Singhand Singh, 2019c).
PFNA decreased protein levels of SF1, a transcription factor involved in sex
differentiation that regulates transcription of StAR and CYP11a, in prepubertal males
exposed for 14 days to 2 or 5 mg/kg-day (Singh and Singh 2019b) and in neonatal
males examined on PND 3 after in utero exposure at 5 mg/kg-day (Singh and Singh
2019d).
PFNA decreased protein levels of StAR in prepubertal males exposed for 14 days at 2
or 5 mg/kg-day (Singh and Singh 2019b) and in neonatal males examined on PND 3
after in utero exposure at 2 or 5 mg/kg-day (Singh and Singh 2019d), and decreasedmRNA levels in prepubertal males exposed for 90 days at 0.5 mg/kg-day (Singh andSingh 2019a).
PFNA decreased protein levels of CYP11a (P450scc), 3β-HSD, and 17β-HSD in
prepubertal males exposed for 14 days at 2 or 5 mg/kg-day (Singh and Singh 2019b)
and in neonatal males examined on PND 3 after in utero exposure at 5 mg/kg-day(Singh and Singh 2019d), and decreased mRNA levels of CYP11a and 3β-HSD in
prepubertal males exposed for 90 days at 0.5 mg/kg-day (Singh and Singh 2019a).

In male zebrafish, PFNA exposure for 180 days increased gonadal gene expression of star (at0.1 and 1.0 mg/L), cyp11a (at 1.0 mg/L), 17β-hsd (at 0.1 mg/L), and cyp19a (at 0.1 mg/L),
reduced gonadal gene expression of 3β-hsd (at 0.01, 0.1, and 1.0 mg/L), and had no effect on
gene expression of cyp17 (Zhang et al. 2016).
No effects on aromatase activity were observed in a human placental choriocarcinoma cell line
(JEG-3 cells) following treatment with PFNA (Kjeldsen and Bonefeld-Jørgensen 2013).
In a mouse Leydig tumor cell line (mLTC-1 culture), incubation with PFNA inhibitedprogesterone production with an IC50 value of 16.6 (Zhao et al. 2017). The authors suggested
that PFNA disrupted progesterone production as a result of damage to the mitochondrial
membrane and increased production of reactive oxygen species (ROS).
Hormone receptor mediated effects have been studied in vivo in mice and fish, and in vitro in ahuman embryonic kidney cell line, two human breast adenocarcinoma cell lines, cultured
primary mouse Sertoli cells, and a Chinese hamster ovary cell line after exposure to PFNA. In
addition, hormone receptor binding studies have been conducted with fish liver cytosol andmodeled in silico.
In prepubertal mice, PFNA treatment for 14 days reduced testicular gene expression of several
hormone receptors, specifically mRNAs for androgen receptor (AR), growth hormone receptor(GHR), and IGF-1R at doses of 2 and 5 mg/kg-day, and IGF-1 at 5 mg/kg-day (Singh andSingh 2019c).

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In male zebrafish, PFNA treatment increased gene and protein expression of vtg, an estrogenresponsive gene, in the liver (Zhang et al. 2016). In this study PFNA treatment also alteredgene expression in male zebrafish of certain hormone receptors in the brain (decreased mRNAlevels for era, erb, and ar), gonads (decreased mRNA levels for fshr and lhr) and liver(increased mRNA levels for era and erb). In rainbow trout, PFNA treatment via diet resulted inconcentration-dependent increases in plasma levels of Vtg in studies with 11-month oldjuvenile trout (Benninghoff et al. 2011). In other experiments by these authors, PFNA wasshown to competitively bind (very weakly) to trout liver Er (in liver cytosol homogenates)(Benninghoff et al. 2011).
In human embryonic kidney (HEK293T) cells transfected with a human ERα reporter gene,
PFNA (100-1000 nM) increased human ERα (hERα) reporter activity up to 2.5 fold
(Benninghoff et al. 2011).
In MVLN cells (derived from MCF-7 cells, a human breast adenocarcinoma cell line, stablytransfected with a luciferase reporter gene and an estrogen-responsive element from the
Xenopus vitellogenin A2 gene), exposure to PFNA alone did not induce an estrogenic
response. However, PFNA displayed anti-estrogenic activity in MVLN cells pretreated with E2,
inhibiting the estrogenic response to E2 (measured as luciferase activity) in a concentration
dependent manner. PFNA was a less potent inhibitor of the estrogenic response than PFDA in
these cells, with an EC50 of 28.1 µM (Juan Li et al. 2020). In separate experiments in MCF-7
cells (a human breast adenocarcinoma cell line), PFNA also had anti-estrogenic activity, withthe addition of PFNA to E2-treated cells resulting in significant down regulation of the E2responsive genes trefoil factor 1 (TFF1, also known as pS2) and EGR3 (early growth response3) (Juan Li et al. 2020).
In primary rat Sertoli cell cultures, incubation with PFNA for 24 hours had no effect on mRNAlevels of AR at concentrations ranging from 1 to 75 µM, increased levels of androgen binding
protein (ABP) at ≥50 µM, reduced levels of transferrin mRNA (involved in spermatogenesis) at≥25 µM, and reduced mRNA levels of FSH-R at 25 and 50 µM (Feng et al. 2010).
PFNA significantly antagonized DHT-induced AR activity in a concentration-dependent mannerin a Chinese hamster ovary cell line (CHO-K1) transfected with AR and a reporter vector(Kjeldsen and Bonefeld-Jørgensen 2013).
Analyses using in silico computational models indicate that PFNA can efficiently dock withhuman, mouse, and trout ERα in the ligand-binding domain and form a hydrogen bond atresidue Arg394 in human ERα in a manner similar to that of estrogens (Benninghoff et al.
2011). Additional in silico modeling studies with the activated form of hERα, in which E2 isbound to the ligand-binding domain, predict that PFNA can bind to sites on the surface of theactivated receptor (Juan Li et al. 2020).

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Table 4.3.1 PFNA: Effects on the hypothalamic-pituitary-gonadal-(liver) axis in animals and in vitro studies 3.
Reference; Experimental Model; N/group;
Exposure Details

Feng et al. 2009

HPG Related Outcomes
Assessed

Serum levels of T, E2, FSH, and LH.

Male SD rats, aged 8 weeks at the beginning of treatment; six animals pergroup.

No effect on serum levels of FSH or LH.

Feng et al. 2010

In vivo: Serum levels of MIS and inhibin B.

In vivo: Male SD rats, 8 weeks old, 6 rats per group.

Testicular levels of WT1 and transferrin

Treatment: PFNA (97% purity) in water (containing 0.2% Tween-20), daily
gavage for two weeks at 0, 3, or 5 mg/kg-day.

In vitro: mRNA levels for MIS, inhibin B, ABP,
FSH-R, AR, WT1, and transferrin

In vitro: Primary cultured rat Sertoli cells
Sertoli cells isolated from 8 week old SD rats were cultured for 3 days andthen treated with PFNA (1, 10, 25, 50, or 75µM) for 24h.
Control: 0.01% DMSO.

Male Wistar rats. PFNA (purity not stated) dosed at:
0.0125 (low, n=10),
0.25 (mid, n=8), or
5 (high, n=8) mg/kg bw/day orally by gavage for a 14-day with corn oil
vehicle control (n = 10

Increased serum T level (by 87.5%) at 1 mg/kg-day, reduced (by 85.4%)
at 5 mg/kg-day.
Increased E2 at 5 mg/kg-day.

Treatment: PFNA (97% purity) in water (containing 0.2% Tween-20), daily
gavage for two weeks at doses of 0, 1, 3, or 5 mg/kg-day.

Hadrup et al. 2016

Results

Plasma levels of androstenedione, T, DHT,
LH, FSH and prolactin.
mRNA levels of 17β-HSD, StAR,
benzodiazepine receptor (BZRP; i.e.,
mitochondrial translocator protein or TSPO),CYP11A, and CYP17 in testicular tissues.
body weight

In vivo:
Increased serum levels of MIS at 5 mg/kg-day
Reduced serum levels of inhibin B at all doses.
Increased testicular levels of WT1 at 5 mg/kg-day
Decreased testicular levels of transferrin at all doses.
In vitro
Increased MIS mRNA levels at 10, 25, 50, or 75 µM
Reduced inhibin B mRNA levels at 50 and 75 µM
Increased levels of ABP mRNA at 50 and 75 µM
Reduced FSH-R mRNA levels at 25 and 50 µM.
No effects on mRNA levels of AR.
Increased WT1 mRNA levels at all doses.
Decreased transferrin mRNA levels at 25, 50 and 75 µM
The authors stated that plasma levels of androstenedione andtestosterone were decreased at 5 mg/kg-day, but no statisticallysignificant differences from control levels were noted in Figure 3. No
effect on plasma DHT. The authors did not mention differences fromcontrol in plasma LH, FSH or prolactin levels associated with PFNA
treatment; however, Figure 4 suggests that LH may be reduced and FSH
and prolactin may be increased at 5 mg/kg-day.
No effect of 0.0125 or 0.25 mg/kg-day on testicular mRNA levels of 17βHSD. At 5 mg/kg-day, testicular mRNA levels of 17β-HSD, StAR, BZRP(TSPO), CYP11A, and CYP17 were decreased (data were not shown).
Decreased body weight at high dose (5 mg/kg-day)Studies with in vivo experiments are presented first ordered by species (rat, mouse, fish), and within species, by publication date, followed bystudies with only in vitro experiments, ordered by publication date.
3

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Reference; Experimental Model; N/group;
Exposure Details

NTP 2019

HPG Related Outcomes
Assessed

Results

Serum T levels
Survival, body weight

Reduced serum T at 2.5 mg/kg-day

Singh and Singh 2019a

Serum T levels.

Male Parkes mice, 25 days old. 14 animals per group.

Analysis of mRNA expression bysemiquantitative RT-PCR: StAR, CYP11A1,
3β‐HSD, 17β‐ HSD.
Immunoblotting: StAR, CYP11A1, 3β‐HSD,
17β‐ HSD.

At 0.2 mg/kg-day: No effect on serum T level or expression of genes orproteins assessed.
At 0.5 mg/kg-day:Reduced serum T level (31%).
Down-regulation in testicular mRNA levels of StAR, CYP11A1, 3β‐HSD
and 17β‐ HSD. Decreased testicular protein levels of StAR, CYP11A1,
3β‐HSD, and 17β‐ HSD.

Body weightNo effect on body weight.

Singh and Singh 2019b

Serum and intratesticular levels of T.

Male Parkes mice, 25 days old. 10 animals per group.

Protein levels by immunoblotting: SF1, StAR,
CYP11A1, 3β-HSD, 17β- HSD.

Decreased serum T level by 72% and 85%, at 2 and 5 mg/kg-day,respectively, and decreased intratesticular T at both doses.

Male SD rats, 9-11 weeks old at the beginning of dosing, 10 rats per dosegroup.

Decreased survival in the 5 and 10 mg/kg-day dose groups (2/10 and
0/10, respectively). Lower body weights at ≥ 1.25 mg/kg-day.

Treatment: PFNA (99.5% purity) once daily by gavage in deionized waterwith 20% Tween® 80 at doses of 0, 0.625, 1.25, 2.5, 5.0 or 10.0 mg/kg-day,respectively, for 28 days.

Treatment: PFNA (97% purity) in distilled water, daily gavage at 0, 0.2, or
0.5 mg/kg-day, from PND 25 to 114 (for 90 days). The animals weresacrificed on PND 115.

Treatment: PFNA (97% purity) in distilled water, daily gavage at 0, 2, or 5
mg/kg-day from PND 25 to 38. The animals were sacrificed on PND 39.
Singh and Singh 2019c[Study design same as the 2019b study by the same authors.]
Male Parkes mice, 25 days old. 10 animals per group,

Body weight.

Decreased protein levels of SF1, StAR, CYP11A1, 3β-HSD, and 17βHSD at both doses.
Reduced body weight gain (24%) at 5 mg/kg-day.

Testicular mRNA levels for AR, GHR, IGF-1,
insulin like growth factor 1 receptor (IGF-1R)
and HGM Co Synthase 1

Reduced mRNA expression of AR, GHR, and IGF-1R at both doses
Decreased mRNA expression of IGF-1 and HGM Co Synthase 1 at 5
mg/kg-day.

Treatment: PFNA (97% purity) in distilled water, daily gavage at 0, 2, or 5
mg/kg-day from PND 25 to 38.

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Reference; Experimental Model; N/group;
Exposure Details

HPG Related Outcomes
Assessed

Results

Female Parkes mice, 10 pregnant mice per group. For testis evaluation, 2male pups from each dam (a total of 20 pups per group) were sacrificed on
PND 3.

Evaluation of testicular tissue from male pups:
Immunohistochemistry: 3β‐HSD

Reduction in intratesticular levels of T at 5 mg/kg-day(1.07 ± 0.07 ng/g tissue vs 1.83 ± 0.105 ng/g tissue in controls)

Immunoblotting: StAR, CYP11A1, 3β‐ HSD,
17β‐HSD

Treatment: PFNA (97% purity) in distilled water, daily gavage to pregnant
mice at 0, 2, or 5 mg/kg-day from GD 12 until parturition.

Gene expression of anti-mullerian hormone(Amh, MIS).

Decreased protein levels of StAR at both doses, and of CYP11A1, 3β‐
HSD, 17β‐HSD, and SF1 at 5 mg/kg-day. Reduced 3β‐HSD protein was
also found by immunohistochemical staining.

Singh and Singh 2019d

One testis from each of five male pups per group was processed forhistology and immunohistochemistry. The remaining testes from these 5
pups and all other 15 male pups were used for immunobotting and othermeasurements.

Decreaed MIS mRNA at 2 mg/kg-day, and increased mRNA at 5 mg/kgday.

Benninghoff et al. 2011

In Vivo

In Vivo

In vivo: Rainbow trout

Vtg levels in plasma

Concentration dependent increase in plasma Vtg levels

In Vitro

In Vitro

Dietary exposure of 11-month old juvenile rainbow trout to PFNA (highest
purity available) at
0, vehicle control (DMSO), 5, 50, or 250 ppm in Oregon Test Diet, OTD) 5
days/week for 14 days (equivalent to 0.1, 1, or 5 mg/kg-d, respectively);
animals were sacrificed on day 15. Positive control: 5 ppm E2

Trout liver cytosol: ERα binding studies

Trout liver cytosol: PFNA shows weak competitive binding to ERα,
completely displacing E2 from the receptor, with more than a 10,000-foldlower relative binding affinity than E2.

HEK-293T cells: hERα gene reporter activity

HEK-293T cells: PFNA at concentrations of 100-1000 nM induced hERαgene reporter activity up to 2.5 foldIn vitro: trout liver cytosol, HEK-293T cells

In Silico

In Silico

Trout liver cytosol

Molecular docking at the active site in human,
mouse and trout ERαPFNA is predicted to bind at the active site of human, mouse, and trout
ERα. Predicted hydrogen bond interaction with hERα at Arg394.

N = 6 fish/treated and vehicle control (0.5 ppm DMSO) group.
N = 24 fish in untreated control group

ER binding studies in liver cytosol prepared from trout fed 5 ppm E2 (toinduce ERα)HEK-293T cells: a human embryonic kidney cell line transfected with ahuman ERα expression vector and a luciferase reporter plasmid containing
an estrogen response element (ERE).
PFNA or E2 treatment (1-1000 nM) for 24 hrs; hERα reporter gene activity
measured.
In Silico
Molecular docking studies of PFNA with the active site in human, mouse,
and trout ERα.

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Reference; Experimental Model; N/group;
Exposure Details

HPG Related Outcomes
Assessed

Zhang et al. 2016

Serum levels of T and E2.

Zebrafish (wild type, Tuebingen strain): five-month-old (n = 480)). Animals
were separated by sex and randomly assigned to treatment groups

Vitellogenin mRNA (vtg) and protein (Vtg)
levels in male liver.

Treatment: PFNA (97% purity) in water through a flow-through exposuresystem at concentrations of 0, 0.01, 0.1 and 1.0 mg/L for 180 days.
Serum hormone measurements were performed on 3 pooled samples(pooled from 20 males) per dose group. mRNA analyses were performed on
6 males per dose group.

Transcriptional profiles of genes associatedwith the synthesis of sex hormones and the
hypothalamic-pituitary-gonadal-liver axis
(HPGL axis):
Brain: estrogen receptor α (era), estrogen
receptor β (erb), ar, cytochrome P450 family
19 subfamily B (cyp19b), luteinizing hormone
β (lhb).
Gonad: fshr, lhr, star,
cyp11a, 3β-hsd, cyp17, 17β-hsd, cyp19a.
Liver: era and erb.

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53

Results
Increased serum T at 0.01 mg/L in males (and females). Increased serum
E2 at 0.1 and 1.0 mg/L in males (and females). The T/E2 ratio in males
from the 0.01 and 0.1-mg/L groups appeared to be increased(approximately 180 at 0.01 mg/L and 110 at 0.1 mg/L vs. 45 in controls),
but statistical significance was not indicated.
Increased vtg mRNA levels in male liver at all dosing levels.
Increased Vtg protein levels in male liver at 0.1 and 1.0 mg/L.
Altered mRNA levels of genes related to the HPGL axis in males:Brainera: decreased at 0.01 mg/L, increased at 0.1 mg/Lerb: decreased at
0.01 mg/L, increased at 0.1 mg/L
ar: decreased at 0.1 and 1.0 mg/L
cyp19b: no change
fshb: no change
lhb: increased at 0.1 mg/L
Gonad
fshr: decreased at 0.1 and 1.0 mg/L
lhr: decreased at all doses
star: increased at 0.1 and 1.0 mg/L
cyp11a: increased at 1.0 mg/L
3β-hsd: decreased at all doses
cyp17: no change
17β-hsd: increased at 0.1 mg/L
cyp19a: increased at 0.1 mg/L
Liverera: increased at 0.1 and 1.0 mg/L
erb: increased at 0.1 mg/L

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Reference; Experimental Model; N/group;
Exposure Details

HPG Related Outcomes
Assessed

Results

Kjeldsen and Bonefeld-Jørgensen 2013

MVLN cells: ER transactivation

MVLN cells

In vitro: MVLN, CHO-K1 and JEG-3 cell lines

CHO-K1 cells: AR transactivation

Cytotoxic at 10-4 M

PFNA (97% purity) dissolved in dimethyl sulfoxide (DMSO), tested atconcentrations of 10-9 – 10-4 M

JEG-3 cells: Aromatase activity

MVLN cells: Derived from MCF-7 cells; stably transfected with a luciferase
reporter gene and an estrogen responsive element derived from the
Xenopus vitellogein A2 gene.

ER transactivation: No effects on ER activity
CHO-K1cells
Cytotoxic at 10-4 M
AR transactivation: No AR agonist activity.
Concentration-dependent antagonistic effects on DHT-induced AR
transactivation (IC50 5.2×10-5 M).

ER transactivation assessed with PFNA alone or co-treated with 25 pM E2
CHO-K1 cells: Derived from CHO cells; transiently co-transfected with theAR plasmid and a luciferase reporter vector

JEG-3 cells
Cytotoxic at 10-4 M

AR transactivation assessed with PFNA alone or co-treated with or 25 pM
DHT

No effects on aromatase activity.

JEG-3 cells: A human choriocarcinoma cell line. Aromatase activity
assessed with PFNA treatment
Zhao et al. 2017

Progesterone production

In vitro: Mouse Leydig cell tumor line (mLTC-1)

Mitochondrial membrane potential

PFNA (>95% purity)
Experiment 1: PFNA treatment (0-105µM) for 24 hrs; cAMP added andincubated for additional 3 hrs; progesterone quantified via
radioimmunoassay

Experiment 1: Concentration dependent decrease in progesterone
production (IC50 16.61 µM)
Experiment 2: Significant decrease in mitochondrial membrane potential
at 50 and 100 µM.

Experiment 2: PFNA treatment (0, 12.5, 25, 50 or 100 µM) for 24 hours;
mitochondrial membrane potential measured

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Reference; Experimental Model; N/group;
Exposure Details

Juan Li et al. 2020

In vitro: MVLN and MCF-7 cell lines
PFNA (97% purity)
MVLN cells: Derived from MCF-7 cells; stably transfected with a luciferase
reporter gene and an estrogen responsive element derived from the
Xenopus vitellogein A2 gene.

HPG Related Outcomes
Assessed

MVLN cells: ER transactivation
MCF-7 cells: mRNA gene expression of
estrogen regulated genes TFF1/pS2, EGR3
In silico: modeling predictions of interaction
with E2-activated hERαPFNA treatment (0, 0.0001-5 mM) for 48 hr in the presence or absence ofE2 (1 nM) as positive control or ICI 182,780 as negative control, or both; ER
transactivation

Results
MVLN cells
ER transactivation: No ER agonist activity with PFNA alone; in cells
pretreated with E2 PFNA inhibited the estrogenic response to E2 in aconcentration dependent manner, with a reported EC50 of 28.1 µM.
In cells cotreated with E2 and ICI 182,780 , PFNA caused a significant
increase in the inhibition of luciferase activity (attenuated luciferase
response)
MCF-7 cells
mRNA levels of TFF1 and EGR3: No effect on gene expression with
PFNA alone. In cells cotreated with E2, PFNA significantly
downregulated expression of both TFF1 and EGR3

MCF-7 cells: human breast adenocarcinoma cell line
PFNA treatment (50 µM) in the presence or absence of E2 for 48 hours;changes in expression of estrogen regulated genes (TFF1/pS2, EGR3)

In silicoModeling predicts that PFNA can bind to the surface of the E2 activatedform of hERα (at sites distinct from the ER binding pocket)

In silicoModeling analysis of PFNA interactions with E2-activated hERαPFNA and its salts andPFDA and its salts

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Effects on the thyroid
PFNA effects on thyroid hormones are summarized in Table 4.3.2 below.
A subset of data from the National Health and Nutrition Examination Survey (NHANES)
provided a cross-sectional look at serum levels of PFNA, thyroid hormones, andtestosterone in human males aged from 12-80 years (Lewis et al. 2015). Only one
whole-animal study in rodents (NTP 2019) provided data on outcomes of both thyroid
toxicity and male reproductive toxicity with repeated exposures to PFNA. The only
additional whole-animal study of thyroid hormone relevant outcomes involved repeated
PFNA dosing of two generations of zebrafish, but did not report on reproductive
parameters (Y Liu et al. 2011). An in vitro study using rat pituitary GH3 cells evaluated
PFNA for cytotoxicity, effects on cell proliferation, and aryl hydrocarbon receptor (AhR)
transactivity (Long et al. 2013). Two studies of PFNA involved cell-free biochemical
assays of thyroid binding (Ren et al. 2016; Weiss et al. 2009). The Ren (2016)
publication included an in silico molecular docking simulation to model binding of PFNA
to thyroid hormone transport proteins.
The Lewis et al. (2015) study analyzed serum samples from a total of 857 human males
divided into age groups. Median PFNA levels increased with age from 0.78 ng/mL for 12
to <20 year-olds, to 1.07 ng/mL for individuals aged 60 – 80 years. Samples were also
analyzed for levels of testosterone, total TSH, free and total T3 and T4, and several
other PFASs. Statistical modeling of percent change in hormone concentration per
doubling of PFNA provided only one statistically significant result: a 16.3% increase in
TSH for 12 to <20 year-olds. A non-significant increase in total T4 was determined forsampled 40-60 year olds. Adjustments were made for several covariates, but not forother PFASs in the samples.
The NTP (2019) 28-day rat study found that excess mortality at the two highest
administered doses of PFNA (5 and 10 mg/kg-day) precluded further assessment ofoutcomes for those groups. As a result, 2.5 mg/kg-day was the effective high dose forreported outcomes. There was no significant effect on T3. Statistically significant
outcomes relevant to thyroid toxicity included:
•
•
•
•
•

Decreased absolute thyroid gland weight at 2.5 mg/kg-day
Increased relative thyroid weight at 2.5 mg/kg-day
Decreased TSH at doses > 1.25 mg/kg-day
Decreased total T4 at 0.625 and 1.25 mg/kg-day (not 2.5 mg/kg-day)Decreased free T4 at doses > 0.625 mg/kg-day

The same animals showed decreased testosterone levels at 2.5 mg/kg-day. Adverse
male reproductive outcomes observed at doses > 1.25 mg/kg-day included reduced
testes and epididymis weights, as well as multiple histopathological changes in testes.

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Two generations of zebrafish were continuously exposed to PFNA at concentrations of0, 0.05, 0.1, 0.5, and 1 mg/l (Y Liu et al. 2011). While F0 males and females were sexed
at maturity, and then paired for spawning the F1, no mention was made of sex ratios or
fertility of these animals.
Reported outcomes included:
•
•
•
•
•
•
•
•

Significantly elevated plasma T3 levels in both F0 and F1 adult males.
T3 + T4 was significantly higher in F0 adult males than in controls.
PFNA-induced histological changes in F0 male zebrafish indicative ofhyperstimulation of thyroid follicles.
Significantly increased hepatic levels of transcript for transthyretin (TTR) in F0
males.
In samples of brain tissue from F0 animals, TRα transcription was downregulated at 0.1 and 0.5 mg/l; TRß was down-regulated at 0.1 mg/l.
In homogenized F1 larvae, expression of TRα was induced at concentrations> 0.1 mg/l; TRß was induced at all PFNA concentrations tested.
TTR expression was increased in F1 larvae following parental exposure to PFNA
at concentrations of 0.05 mg/l or greater.
Overall gene expression profile studies indicated PFNA-induced disruption of
thyroid hormone transport, metabolism, synthesis and function.

Liu et al. (2011) suggested that observed increased TTR transcription in zebrafish could
reflect induction due to competitive binding of PFNA. The authors propose that PNFA
could induce TTR transcription across species, while producing different results in ratsand zebrafish for thyroid hormone levels.
Weiss et al. (2009) tested PFNA, as well as other related compounds, for binding to thehuman thyroid transport protein TTR in competition with 125I-labeled T4. PFNA inhibited
18% of T4-TTR binding. Binding potency among the series of tested compounds
decreased in the order: PFHxS> PFOS/PFOA> perfluoroheptanoic acid (PFHpA)>
sodium perfluoro-1-octanesulfinate > PFNA. TTR binding potencies ranged from 12.5–
50 times lower than T4 (Weiss et al. 2009).
An in vitro study of PFNA effects on proliferation of T3-dependent rat pituitary GH3 cells
found no cytotoxicity under test conditions (Long et al. 2013). Concentration-dependent
decreases in GH3 cell proliferation were seen with PFNA alone (1×10−9–1×10−4 M) orin combination with 0.5 nM T3 (1x10-8-1x10-4). Either alone or in combination with
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PFNA had no effect on AhR
transactivity.
In a cell-free assay, the relative potency for PFNA binding with TTR was 0.016, ascompared to T4 representing a relative potency of 1 and 0.083 for PFOA (Ren et al.
2016). In experiments comparing binding to wild type versus mutant thyroxine-binding
globulin (TBG), arelative potency was not detectable for PFNA binding to wild type TBG.

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In silico molecular docking experiments to TTR and TBG found PFNA to be among thetested perfluoroalkyl acids and sulfonates that fit the binding pockets (Ren et al. 2016).
Overall, experimental evidence from cell-free binding assays and in silico moleculardocking models shows binding of PFNA to TTR (Ren et al. 2016; Weiss et al. 2009) withconsequences of PFNA exposure for thyroid hormone levels and function observed incell culture (Long et al. 2013) and zebrafish (Y Liu et al. 2011). The available humandata (Lewis et al. 2015) found no effect of PFNA on testosterone, and a significant
effect on TSH levels only for boys in the adolescent age-range. Only the NTP (2019)study in rats reported direct evidence of male reproductive toxicity with exposure to
PFNA at doses also associated with decreases in TSH and free T4 (NTP 2019). Thisco-occurrence of effects is consistent with, but neither establishes nor eliminates,
potential for a causal, mechanistic role for thyroid disruption in the male reproductive
toxicity of PFNA.

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Table 4.3.2 PFNA Effects on thyroid hormones.
Study Design

Outcomes Assessed

Major Findings

Comments

NTP 2019 (see Table 4.2.1 forsummary of male repro data from thisstudy)

Body weights and thyroid
gland weights and histology.

Due to excess mortality at doses of 5 or 10 mg
PFNA/kg-day, further analyses were notpresented for outcomes at those doses.

In the same study, direct
evidence of male reproductive
toxicity was reported at doses >1.25 mg/kg-day:Male SD rats, 10-11 weeks old, 10
rats/dose. PFNA administered once
daily by gavage in deionized waterwith 20% Tween® 80 at doses of 0,0.625, 1.25, 2.5, 5.0 or 10.0 mg
PFDA/kg-day, for 28 days.
At the end of the study, blood wascollected from the abdominal aorta forbiochemical analysis (including thyroid
stimulating hormone (TSH) andthyroid hormones).

Blood levels of TSH, T3, andT4 (total and free).

Absolute thyroid gland weight decreased at 2.5
mg/kg-day (p < 0.01).
Increased relative thyroid gland weight at 2.5
mg/kg-day (p < 0.01).
No evidence of follicular cell hypertrophy inthyroid gland.
TSH significantly decreased at 1.25 and 2.5
mg/kg-day (p < 0.01)

Outcomes included reduced
testis and epididymis weights,
reduced serum testosterone
levels, [reduced epididymal
sperm counts, increased
incidence of interstitial tissue
atrophy, germ cell degeneration,and inhibited spermiation.

No significant effect on T3.
Total T4 significantly decreased at 0.625 and1.25 mg/kg-day (p < 0.01) but not at 2.5
mg/kg-day.
Free T4 significantly decreased at doses of
0.625, 1.25, and 2.5 mg/kg-day (p < 0.01).

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Study Design

Outcomes Assessed

Major Findings

Comments

Ren et al. 2016

Binding affinities with thyroid
hormone transport proteins:
TTR and TBG.

The relative potency for PFNA binding with
TTR was 0.016, as compared to T4
representing an RP of 1 and 0.083 for PFOA.

Molecular docking of PFNA
with TTR.

For TBG, binding was not detectable for PFNA
or PFOA.

The molecular docking model
showed that T4 could fill the
TTR ligand-binding pocket.
PFOA and PFOS could also fill
the pocket, with the fluorinatedcarbon tail extended. PFASs
with a medium chain length anda sulfonate acid group were
found to be optimal for TTR
binding, and PFASs with lengths
longer than 12 carbons were
optimal for TBG binding.

Cell-free biochemical assays:
Quantitative assessment of binding
affinities of PFASs (including PFNA,
purity not specified) to humantransthyretin (TTR) and human
thyroxine-binding globulin (TBG) by afluorescent competitive binding assay.

PFNA was one of 16 PFASs shown to fit the
TTR and TBG binding pockets using amolecular docking model.

Molecular docking (in silico) was used
to simulate interaction of PFNA with
TTR.
Lewis et al. 2015

Serum PFNA

Cross-sectional study of U.S. humansubjects

Serum total T

Subset of the National Health andNutrition Examination Survey(NHANES) with serum samples
assayed for PFNA, testosterone, andthyroid hormones.
Data collected in 2011-2012
N = 857 males, 12-80 years

Total TSH
Free and total T3
Free and total T4
Statistical modeling of
associations between PFAS
levels and hormone
concentrations.

Serum PFNA (ng/mL) expressed as median(IQR) by age group:

Adjusted for covariates: age,
BMI, PIR,

12 to <20 years: 0.78 (0.56, 1.19)
20 to <40 years: 0.98 (0.67, 1.31)

race/ethnicity, and serum
cotinine.

40 to <60 years: 1.00 (0.67, 1.57)

Not adjusted for other PFAS:

60 to 80 years: 1.07 (0.77, 1.58)

PFHxS

Percent change (95% CI) in hormone
concentration modeled per doubling of PFNA:

PFOA

Total T4: 40-60-year-olds: -2.5% (-5.2, 0.2),
non-significant apparent association.
TSH: 12 to <20-year-olds: 16.3% (4.0, 30.2), p< 0.05.

Exclusions: missing data on variables
in analyses

PFOS
Correlations among PFASs
were not reported

No associations for other age groups withthese hormones or for any age group withother evaluated hormones.
Long et al. 2013

PFNA and its salts andPFDA and its salts

Cytotoxicity to rat pituitary
GH3 cells and transfectedmouse cells

PFNA was not cytotoxic to rat pituitary GH3
cells or transfected mouse cells under test
conditions.

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Study Design

Outcomes Assessed

Major Findings

Two in vitro tests with PFNA (97%purity):Proliferative response of GH3
cells to PFNA or T3 alone, or
PFNA in the presence of 0.5
nM T3.

Concentration-dependent increase in GH3 cellproliferation with T3 alone at 1×10−10–1×10−8
M.

1. Proliferation of T3- dependent rat
pituitary GH3 cells using the Tscreen assay
2. Effect on aryl hydrocarbon
receptor (AhR) transactivation in
the AhR-luciferase reporter genebioassay in transfected mouse
Hepa1.1 2cR cells.

Effect on AhR transactivity of
PFNA in the absence orpresence of 60 pM TCDD.

Comments

Concentration-dependent decrease in GH3
cell proliferation with PFNA alone at 1×10−9–
1×10−4 M (p < 0.05).
Concentration-dependent decrease in T3induced GH3 cell proliferation with PFNA at
1x10-8-1x10-4 (p < 0.05) in the presence of
0.5 nM T3.
Either alone or in combination with TCDD,PFDA had no effect on AhR transactivity intransfected mouse cells.

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Study Design

Outcomes Assessed

Major Findings

Comments

Y Liu et al. 2011

Plasma thyroid hormone
levels in F0 males after
exposure for 180 days, and in
F1 males at 180 dpf.

Significantly elevated plasma T3 levels in both
F0 (p < 0.05 at 0.05, 0.1, and 1 mg/l PFNA for
180 days) and F1 adult males (p < 0.05 for alldose groups).

Thyroid follicle histology in F0
males after exposure for 180
days.

In F0 males, T4 was elevated above controls
at all PFNA concentrations, but did not reachstatistical significance; T3 + T4 wassignificantly higher than controls at 0.1 and 0.5
mg/l (p < 0.05).

Authors note in discussion
evidence that PNFA could
induce TTR transcription acrossspecies, while producing
different results in rats andzebrafish for thyroid hormone
levels.

Zebrafish (Danio rerio) exposed to
PFNA (97%) at 0, 0.05, 0.1, 0.5, and 1
mg/l. Treatment of F0 started at 23days dpf.
N = 50 fish/concentration group.
Exposure continuous for 180 days.
Sex determined at maturity (120 dpf).
Males and females from the same
dose group were paired for spawning.
F1 offspring raised with or without
continued PFNA exposure for an
additional 180 days.

Gene expression related tothe hypothalamus–pituitary–
thyroid (HPT) axis in F0
males (brain and liver) after
exposure for 180 days, and in
F1 larvae (5 dpf).

PFNA induced histological changes in the
thyroid follicles of F0 males: enlarged follicles
at 0.05 mg/l, hypertrophy of follicularepithelium and follicular cell hyperplasia at 0.1
and 0.5 mg/l; decreased size of follicle lumens
at 1 mg/l.

While F0 males and females
were sexed at 120 dpf, andpaired for spawning the F1, no
mention was made of sex ratios
or fertility of these animals.

Increased gene expression of TTR in F0 liverat 0.1, 0.5, and 1 mg/l (p < 0.05), and in F1 5
dpf larvae at 0.05, 0.5, and 1 mg/l (p < 0.05).
Altered expression of additional HPTassociated genes (e.g., thyroid hormone
transport, metabolism, synthesis and function)
in F0 liver and brain and in F1 5 dpf larvae.
Weiss et al. 2009
Cell-free biochemical assay:
PFNA (97% purity) tested for binding
to human thyroid transport proteintransthyretin (TTR) in competition with
125I-labeled T4.

PFNA and its salts andPFDA and its salts% inhibition of T4-TTR
binding at maximum
concentration, concentration
at 50% inhibition (IC50), slopeof dose-response curve,
relative potency compared to
T4 (T4-REP factor).

For PFNA:
18% inhibition of T4-TTR binding at maximum
concentration
IC50 = 2737 nM (compared to 61 nM for T4)
Slope = -1.11 (compared to -1.17 for T4)
T4-REP factor = 0.022 (T4 = 1)

62

Binding potency of PFASs
decreased in the order: PFHxS> PFOS/PFOA>PFHpA>
sodium perfluoro-1octanesulfinate >PFNA, with
TTR binding potencies 12.5–50
times lower than T4.

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Possible involvement of peroxisome proliferator-activated receptors
Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription
factors that belong to the nuclear hormone receptor family. There are three isotypes of
PPARs, each of which are expressed in rodents and humans: PPARα, PPARβ (alsoknown as δ, NUC-1 or fatty acid activated receptor (FAAR)), and PPARγ. They are
differentially expressed during rat embryonic development and expression levels canfluctuate during different periods of development (Braissant and Wahli 1998). The three
PPAR isoforms are expressed in rat testis, mostly in Leydig and Sertoli cells, and in
human Leydig cell tumors (Kotula-Balak et al. 2020; Michalik et al. 2002). Natural
ligands of PPARs include some fatty acids and eicosanoids. A number of xenobiotic
chemicals are PPAR agonists, including PFNA and other PFASs (e.g. PFDA).
Although PFNA is observed to be an agonist of PPARα (NTP 2019) and PPARγ (C-H Li
et al. 2020), there is little evidence to suggest that PPAR activation is involved in theeffects of PFNA on the testes. The findings from a series of 28-day studies in male rats
conducted by NTP of four perfluoroalkyl carboxylates (perfluorohexanoic acid [PFHxA],PFOA, PFNA, PFDA) and one PPARα agonist (Wyeth-14643) indicate that PPARα
activation is unlikely to be involved in the testicular toxicity observed following treatment
with PFNA (See Section 4.2 and Tables 4.2.1 and 4.2.2) (NTP 2019). Specifically, liver
effects consistent with PPARα activation were observed with Wyeth-14643 and all four
perfluoroalkyl carboxylates, however, only treatment with PFNA or PFDA resulted in
obvious testicular damage, suggesting PFNA and PFDA target the testis through amechanism that does not involve PPARα activation (NTP 2019).
4.4 PFNA: Summary of evidence on male reproductive toxicity
Human studies
The levels of exposure to PFNA in the available epidemiologic studies were low, withmost serum sample medians near 1 ng/mL. The variability in concentrations was also
small, with most IQRs less than 1 ng/mL. These factors would be expected to reduce
the ability of the studies to detect effects associated with PFNA.
The epidemiologic evidence for an effect of PFNA on AGD was mixed, with the strongerof two studies reporting a non-significant association with shorter AGDAS and AGDAPthat did not remain at 12 months of age (Tian et al. 2019).
Three of seven studies observed exposure-related decreases in serum testosterone
levels (two of which were statistically significant) (Cui et al. 2010; Joensen et al. 2013;
Zhou et al. 2016), while the other four studies that presented results for reproductive
hormones reported inconsistent results across locations (Specht et al. 2012) or no

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association between PFNA concentrations and T (Joensen et al. 2009; Lewis et al.
2015; Ma et al. 2021).
Of the six studies that examined effects of PFNA exposure on sperm concentration andcount, five reported no associations (Joensen et al. 2013; Louis et al. 2015; Pan et al.
2019; Petersen et al. 2018; Toft et al. 2012), while a small study with relatively highplasma PFNA concentrations and variability reported a substantial reduction in sperm
concentration and a non-statistically significant reduction in sperm count; thesedifferences did not appear to affect IVF outcomes, such as pregnancies and live births(Ma et al. 2021).
Five of the sperm quality studies examined sperm morphology; of these, one reported
no associations (Joensen et al. 2013) and three reported non-statistically significant
reductions in the proportion of morphologically normal sperm (Ma et al. 2021; Pan et al.
2019; Toft et al. 2012). The fifth study reported an increase in the proportion of normal
sperm (Louis et al. 2015). Pan et al. (2019) also reported that semen PFNA wasassociated with decreased motility, VCL, and VSL.
Sperm DNA integrity was examined in two studies with contrasting population samples:
infertile men were underrepresented in one (Specht et al. 2012), and overrepresented in
the other (Pan et al. 2019). In the latter study, both serum and semen PFNA wereassociated with HDS, and semen PFNA was also associated with DFI. The study bySpecht et al, (2012) also examined markers of apoptosis and reported no consistentassociation with PFNA.
One study examined and found no association between serum PFNA levels andprostate cancer; another examined and found no association between PFNA levels and
PSA concentrations.
Animal studies
PFNA induced dose-dependent reductions in epididymal weight at ≥0.615 mg/kg-day
and testis weight at ≥1.25 mg/kg-day in rats exposed for 28 days (NTP 2019), while inprepubertal mice exposed to PFNA at ≥2 mg/kg-day for 14 days reductions in testis
weight, which did not reach statistical significance, were observed (Singh and Singh2019b).
Histopathological changes in the testis (e.g., germ cell degeneration) were observed inrats in three studies (Feng et al. 2009; Feng et al. 2010; NTP 2019) and in mice in threestudies (Singh and Singh 2019a, 2019b, 2019c). Testicular lesions observed in rats
treated with PFNA by the oral route for 14 days included cell degeneration in
spermatocytes and spermatogonia, and cytoplasmic vacuolization in Sertoli cells (Fenget al. 2009; Feng et al. 2010), while in rats exposed to lower doses of PFNA for 28 days
dose-related increases in germ cell degeneration (≥2.5 mg/kg-day), interstitial cell

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atrophy (≥2.5 mg/kg-day), and spermatid retention (≥2.5 mg/kg-day) were observed(NTP 2019). In prepubertal mice, treatment with PFNA at ≥2 mg/kg-day for 14 days
(Singh and Singh 2019b) or 0.5 mg/kg-day for 90 days (Singh and Singh 2019a)
resulted in increased germ cell degeneration as well as other changes consistent withthese effects (e.g., decreased relative population size of 4C germ cells; decreased
spermatogonial cells in G2 phase).
In studies that measured sperm parameters, a dose-dependent reduction in epididymal
sperm counts was observed in rats treated with PFNA for 28 days (≥ 1.25 mg/kg-day)(NTP 2019), and in mice dose-related reductions in sperm counts, motility and viability
were observed following exposure to PFNA at 0.2 and 0.5 mg/kg-day for 90 days (Singhand Singh 2019a).
Reduced serum levels of T were consistently observed in rats (Feng et al. 2009; NTP
2019) and mice (Singh and Singh 2019a, 2019b) at dosing levels that caused
histopathological lesions or changes in sperm parameters, while increased serum T
levels were observed in the lowest dose group (but not higher dose groups) in one
study in pubertal rats treated for 14 days (at 1 mg/kg-day) (Feng et al. 2009), and in one
study in zebrafish treated for 180 days (at 0.01 mg/L) (Zhang et al. 2016).
Gestational exposure to PFNA at 5 mg/kg-day reduced intratesticular levels of T in miceon PND 3 (Singh and Singh 2019d). In this same study gestational exposure to PFNAat 2 and 5 mg/kg-day reduced testicular protein levels of StAR and WT1, and at 5
mg/kg-day reduced testicular protein levels of SF1, CYP11A1, 3β-HSD, 17β-HSD, and
PCNA (a marker of cell proliferation). These proteins are critical for steroidogenesis
and Sertoli cell function and proliferation in the perinatal period. In another study of malemice, a dose-related delay in preputial separation was observed following gestational
exposures to PFNA at 3.0 and 5.0 mg/kg-day (Das et al. 2015).
Data on the male fertility effects of PFNA are limited to one study in mice, and anotherin zebrafish. In mice, reductions in fertility index and litter size were observed when
PFNA-exposed males were mated with unexposed females (Singh and Singh 2019a).
The design details for the fertility test in this study were not reported. In the zebrafish
study both males and females were exposed to PFNA prior to mating, and reductions inegg production and hatching rate were observed with PFNA treatment (Zhang et al.
2016).
Coherence of results in human and animal studies
In animal studies PFNA exposure reduced sperm counts (rats, mice), sperm motility(mice), and sperm viability (mice), decreased testis (rats, mice) and epididymal (rats)
weights, and induced histopathological damage, including germ cell degeneration andapoptosis (rats, mice). Only one of six epidemiologic studies that measured sperm

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parameters reported a substantial reduction in sperm concentration and a nonstatistically significant reduction in sperm counts associated with PFNA exposure. A
study that measured PFNA in semen reported reductions in motility, VSL, and VCL, butother studies reported no associations with sperm motility.
In animal studies, reduced serum levels of T were consistently observed in rats andmice at dosing levels that caused histopathological lesions or changes in sperm
parameters. Of the seven epidemiological studies that examined effects on T, higher
serum PFNA was significantly associated with lower serum T in three (Cui et al. 2010;
Joensen et al. 2013; Zhou et al. 2016).
Effects of prenatal PFNA exposure on the developing male reproductive system were
examined in both human and animal studies. In animal studies, gestational treatment
with PFNA at 5 mg/kg-day reduced intratesticular levels of T, testicular levels of several
proteins associated with steroidogenesis, and testicular levels of PCNA (a marker of cellproliferation in mice on PND 3 (Singh and Singh 2019d). In another study of male mice
exposed during gestation, PFNA delayed preputial separation (Das et al. 2015). The
findings from two epidemiological studies that examined AGD in boys (assessed at birthand up to 12 months of age) were mixed.
PFNA exposure was associated with decreased fertility index and litter size in the one
mouse study that evaluated endpoints related to male fertility, while in zebrafish PFNA
exposure of both males and females was associated with reductions in egg production
and hatching rate. The one epidemiological study that evaluated endpoints related tomale fertility reported no adverse effects of paternal serum PFNA concentration on IVF
outcomes.
Mechanistic considerations
While a number of mechanistic pathways could potentially contribute to PFNA-induced
male reproductive effects, direct mechanistic relationships to apical outcomes have notbeen established. Potential mechanistic pathways involved in PFNA mediated male
reproductive toxicity include effects on the HPG axis, and effects on thyroid
homeostasis.
HPG axis
PFNA-induced germ cell degeneration, poor sperm quality (reduced production, motility
and viability) in rats and mice, as well as reduced cell proliferation in neonatal mouse
testis following gestational exposure could result from direct insults by PFNA to germ
cells and/or Sertoli cells or via endocrine-mediated pathways (including direct effects on
Leydig cells).

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Whole animal and in vitro studies indicate PFNA affects steroid hormone levels (Feng etal. 2009; NTP 2019; Singh and Singh 2019a, 2019b, 2019d; Zhang et al. 2016). PFNA
reduced plasma levels of T in male rats (Feng et al. 2009; NTP 2019) and mice (Singhand Singh 2019a, 2019b), and intratesticular T levels in mice (Singh and Singh 2019b,
2019d). Effects of PFNA exposure on other hormones include increased serum E2
levels in rats (Feng et al. 2009) and zebrafish (Zhang et al. 2016), increased serum MIS
in rats (Feng et al. 2010), increased MIS mRNA in primary rat Sertoli cells (Feng et al.
2010), altered testicular gene expression of MIS in mice (Singh and Singh 2019d), andincreased brain mRNA levels of the gene encoding LH in male zebrafish (Zhang et al.
2016).
Whole animal studies indicate PFNA affects gene and protein expression of a numberof enzymes and factors involved in steroidogenesis (Singh and Singh 2019a, 2019b,
2019c, 2019d; Zhang et al. 2016) and the findings from one in vitro study are consistent
with an effect of PFNA on hormone synthesis (Zhao et al. 2017). In mice PFNA
decreased testicular mRNA levels of HMG Co synthase 1 (Singh and Singh 2019c),
protein levels of SF1 and StAR (Singh and Singh 2019b, 2019d), mRNA levels of StAR(Singh and Singh 2019a), protein levels of CYP11a, 3β-HSD and 17β –HSD (Singh andSingh 2019b, 2019d), and mRNA levels of CYP11a and 3β-HSD (Singh and Singh
2019a). In zebrafish PFNA also decreased gonadal gene expression of 3β-hsd, but forseveral other steroidogenic enzymes and related factors (i.e., star, cyp11a, 17β-hsd,
cyp19a) PFNA increased gonadal gene expression (Zhang et al. 2016). In a mouse
Leydig tumor cell line, PFNA decreased steroid hormone production, albeit atconcentrations that also reduced mitochondrial membrane potential (Zhao et al. 2017).
There is evidence that PFNA can interact with estrogen receptors from receptor binding
studies (Benninghoff et al. 2011) and this is supported by predictions from in silico
modeling studies (Benninghoff et al. 2011; Juan Li et al. 2020). Estrogen receptor
mediated effects of PFNA have been observed in fish (Benninghoff et al. 2011; Zhang
et al. 2016) and human cell lines (Benninghoff et al. 2011; Juan Li et al. 2020). In fish,
vtg gene and protein expression was increased in zebrafish (Zhang et al. 2016) and Vtg
protein levels were increased in trout (Benninghoff et al. 2011). PFNA induced an
estrogenic response in human embryonic kidney cells transfected with a hERα reporter
gene (Benninghoff et al. 2011), while anti-estrogenic activity was observed in studies
with human breast adenocarcinoma cell lines (Juan Li et al. 2020).
PFNA may also interact with androgen receptors, based on observations of anti-AR
activity in a Chinese hamster ovary cell line (Kjeldsen and Bonefeld-Jørgensen 2013).
One study each in rats, mice, zebrafish and primary rat Sertoli cell cultures indicate that
PFNA may affect gene and protein expression of some hormone receptors, growth
factor receptors, and related proteins. In rats, PFNA decreased testicular protein levelsof transferrin (Feng et al. 2010). In mice, PFNA reduced testicular mRNA levels for AR,

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GHR, IGF-1R, and IGF-1 (Singh and Singh 2019c). In male zebrafish, PFNA
decreased brain mRNA levels for era, erb and ar, decreased gonadal mRNA levels forfshr and lhr, and increased liver mRNA levels for era and erb (Zhang et al. 2016). In
primary rat Sertoli cell cultures PFNA reduced mRNA levels of FSHR, increased mRNAlevels of ABP, and reduced mRNA levels of transferrin (Feng et al. 2010).
Thyroid homeostasis
Binding of PFNA to TTR has been experimentally seen using cell-free binding assays
and in silico molecular-docking models (Weiss et al., 2009; Ren et al., 2016). Effects of
PFNA exposure on thyroid hormone levels and function have been observed in cell
culture (Long et al., 2013) and in zebrafish (Liu et al., 2011). The one available study inhumans evaluating effects of PFNA on both TSH and T (Lewis et al., 2015) found noeffect of PFNA on T, and a significant effect on TSH levels only for boys in theadolescent age-range. In male rats, evidence of male reproductive toxicity was reportedwith exposure to PFNA at doses also associated with decreases in TSH and free T4(NTP, 2019). The co-occurrence of effects on thyroid hormone and male reproductive
outcomes following PFNA exposure is consistent with, but cannot confirm, a causal,
mechanistic role for thyroid disruption in the male reproductive toxicity of PFNA.
Key characteristics of male reproductive toxicants and endocrinedisrupting chemicals
Recently a set of key characteristics (KCs) that are frequently exhibited by exogenous
agents that cause male reproductive toxicity was identified, based on a survey of known
male reproductive toxicants and established mechanisms and pathways of toxicity(Arzuaga et al. 2019). The KCs can encompass many types of mechanistic endpoints
and are not constrained to previously formulated hypotheses, allowing a broader
consideration of multiple mechanistic pathways and hypotheses. The eight KCs of male
reproductive toxicants are presented in Table 4.4.1. A chemical need not exhibit all of
the KCs, and is not expected to exhibit all of the KCs, in order to be considered a male
reproductive toxicant.
Another set of KCs has been developed for endocrine-disrupting chemicals (EDCs),
based on knowledge of hormone actions and endocrine-disrupting chemical effects (LaMerrill et al. 2020). Because endocrine-disrupting chemicals may be male reproductive
toxicants, the set of ten KCs of endocrine-disrupting chemcals are presented in Table
4.4.2. As is true for the KCs of male reproductive toxicity, a chemical need not exhibit
all of the KCs, and is not expected to exhibt all of the KCs, in order to be considered an
endocrine-disrupting chemical.

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Table 4.4.1 Key characteristics of male reproductive toxicants
Key characteristic

Example of relevant evidence

1. Alters germ cell
development, function, or
death

Increased germ cell apoptosis; alterations in sperm acrosome
reaction and motility

2. Alters somatic cell
development, functions, or
death

Increased Sertoli cell apoptosis; alterations in Sertoli cellfunctions, cytoskeleton, and interactions with germ cells;
alterations in Leydig cell development

3. Alters production andlevels of reproductive
hormones
4. Alters hormone receptor
levels/functions

Decreased Leydig cell steroidogenic functions; increased
hepatic metabolism and excretion of sex hormones
Androgen receptor antagonism, estrogen receptor activation,
decreased LH receptor expression

5. Is genotoxic

DNA damage, chromosome fragmentation, altered sperm cellchromosome numbers

6. Induces epigenetic
alterations

Altered sperm noncoding RNA (ncRNAs), germ cell DNA
methylation patterns, and histone retention sites

7 Induces oxidative stress

Reduced tissue antioxidant levels

8. Induces inflammation

Increased testicular expression of pro-inflammatory markers
and prostaglandin levels

Source: (Arzuaga et al. 2019)PFNA and its salts andPFDA and its salts

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Table 4.4.2 Key characteristics of endocrine-disrupting chemicals
Key characteristic

Example of relevant evidence

1. Interacts with or activates hormone
receptors

Binding or agonism of hormone receptors

2. Antagonizes hormone receptors

Antagonism of nuclear or cell surface hormone
receptors

3. Alters hormone receptor expression

Abundance, distribution and degradation of hormone
receptors

4. Alters signal transduction inhormone- responsive cells factors andtranscripts, and activity

Abundance of post- translational modifications,cofactors, transcription

5. Induces epigenetic modifications inhormone- producing or hormone
responsive cells

Chromatin modifications, DNA methylation and noncoding RNA expression

6. Alters hormone synthesis

Expression or activity of enzymes or substrates inhormone synthesis

7. Alters hormone transport acrosscell membranes

Intracellular transport, vesicle dynamics or cellularsecretion

8. Alters hormone distribution or
circulating hormone levels

Blood protein expression and binding capacity , bloodlevels of pro- hormones and hormones

9. Alters hormone metabolism or
clearance of hormones

Inactivation, breakdown, recycling, clearance,
excretion or elimination

10. Alters fate of hormone- producing
or hormone-responsive cells

Atrophy , hyperplasia, hypertrophy , differentiation,
migration, proliferation or apoptosis

Source: (La Merrill et al. 2020)

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The available mechanistic information on PFNA is discussed below in relation to the KCs formale reproductive toxicants and the KCs for EDCs.
The available studies on PFNA and its salts provide evidence related to three KCs of male
reproductive toxicants, and six KCs of endocrine disrupting chemicals. This information isbriefly summarized below. There is clear overlap among KC 3 of male reproductive toxicants(Male KC3) and KC8 of endocrine-disrupting chemicals (EDC KC 8), where both refer to aneffect on hormone levels, with Male KC3 being specific to effects on reproductive hormone
levels. The data relevant to effects on reproductive hormone levels are therefore presented as
being related to both of these KCs.
Male KC1. Alters germ cell development, function, or death
The findings from human studies of PFNA and its potential to cause effects in sperm were notconsistent. An association with reduction in sperm concentration and a non-statisticallysignificant reduction in sperm count was reported (Ma et al. 2021); but no associations werenoted in other studies (Joensen et al. 2013; Louis et al. 2015; Pan et al. 2019; Petersen et al.
2018; Toft et al. 2012). Reduction in the proportion of morphologically normal sperm with
reduced motility was noted (Pan et al. 2019) though a reduction in the proportion ofmorphologically normal sperm noted in another study was not statistically significant (Toft et al.
2012). An increase in the proportion of normal sperm was also reported (Louis et al. 2015).
A number of animal studies have shown effects of PFNA either on sperm paramenters or
testicular histopathology. Reduction in epididymal sperm counts were noted in rats (NTP2019), and in mice, dose-related reductions in sperm counts, motility and viability wereobserved following exposure to PFNA (Singh and Singh 2019a). Histopathological changes inthe testis with germ cell degeneration were observed in rats (Feng et al. 2009; Feng et al.
2010; NTP 2019) and in mice (Singh and Singh 2019a, 2019b, 2019c).
Male KC2. Alters somatic cell development, functions, or death
Studies in animals have reported inhibited proliferation of Sertoli cells, as evidenced by
reduced protein level of PCNA in testicular tissues, in neonatal mice exposed gestationally to
PFNA (Singh and Singh 2019d).
Male KC3: Alters production and levels of reproductive hormones;
EDC KC8: Alters hormone distribution or circulating hormone levels
Some studies in humans observed exposure-related decreases in serum testosterone levels(two of which were statistically significant) (Cui et al. 2010; Joensen et al. 2013; Zhou et al.
2016), while other studies that presented results for reproductive hormones reported

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inconsistent results across locations (Specht et al. 2012) or no association between PFNA
concentrations and T (Joensen et al. 2009; Lewis et al. 2015; Ma et al. 2021). One study inhumans measured SHBG, and reported that a decrease in SHBG was associated with PFNA
levels in semen and serum (Cui et al. 2020). A number of animal studies have shown effects
on reproductive hormones. Reduced serum levels of T in rats (NTP, 2019; Feng et al. 2009)and mice (Singh and Singh 2019a, 2019b) were observed at dosing levels that caused
histopathological or sperm changes. Increased serum T levels were observed in the lowest
dose group (but not higher dose groups) in pubertal rats (Feng et al. 2009), and in zebrafish(Zhang et al. 2016). Gestational exposure to PFNA reduced intratesticular levels of T in miceon PND 3 (Singh and Singh 2019d).
Effects of PFNA exposure on other hormones include increased serum E2 levels in rats (Fenget al. 2009) and zebrafish (Zhang et al 2016), increased serum MIS in rats (Feng et al. 2010)
and decreased steroid hormone production, at concentrations that also reduced mitochondrial
membrane potential (Zhao et al. 2017).
Male KC4. Alters hormone receptor levels/functions and
EDC KC2. Antagonizes hormone receptors and EDC KC4.
Studies are available on PFNA and its potential to affect hormone receptors. PFNA has beenshown to increase hERα reporter activity in HEK293T cells (Benninghoff et al. 2011); haveantagonistic effects on DHT-induced AR transactivation in CHO-K1 cells (Kjeldsen andBonefeld-Jørgensen 2013); supress luciferase induction by E2 (in a concentration responsemanner) in MVLN cells, and cause significant down regulation in levels of estrogen responsive
genes in these cells (Juan Li et al. 2020).
Male KC5. Is genotoxic
The literature search identified one study in humans and one in vitro animal study relevant tothis key characteristic. In the human study, semen and serum PFNA were associated withsmall increases in high DNA stainability (Pan et al. 2019). In the in vitro study, PFNA caused
an increase in DNA damage, as measured by the comet assay, in testicular cells fromseminiferous tubules in Wistar rats (Lindeman et al. 2012).
EDC KC1. Interacts with or activates hormone receptors
Studies suggest that PFNA interacts with the ER. PFNA showed weak competitive binding to
the ER in trout liver cytosol homogenates (Benninghoff et al. 2011), and predictions from insilico modeling studies are consistent with PFNA interacting with human, mouse and trout ER(Zhang et al. 2016; Benninghoff et al. 2011). Studies have also reported ER mediated effects
of PFNA in fish (Zhang et al. 2016; Benninghoff et al. 2011) and a human cell line (Benninghoff

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et al. 2011). In a MVLN cell line, suppression of luciferase induction by E2 in a concentrationresponse manner was observed, suggesting PFNA interacts with the ER (Juan Li et al. 2020).
EDC KC2. Antagonizes hormone receptors.
In CHO-K1 cells, PFNA caused a concentration dependent antagonistic effect on DHT-induced
AR transactivation (Kjeldsen and Bonefeld-Jorgensen 2013) and in MVLN cells, PFNA
suppressed luciferase induction by E2 in a concentration-response manner, suggesting that
PFNA can inhibit the ER (Juan Li et al. 2020).
EDC KC3. Alters hormone receptor expression
Studies in mice, zebrafish and primary rat Sertoli cell cultures report PFNA-induced changes ingene and protein expression of some hormone receptors, growth factor receptors, and related
proteins. In mice, PFNA reduced testicular mRNA levels for AR, GHR, IGF-1R, and IGF-1(Singh and Singh 2019c). In male zebrafish, PFNA decreased brain mRNA levels for era, erb
and ar, decreased gonadal mRNA levels for fshr and lhr, and increased liver mRNA levels forera and erb (Zhang et al. 2016). In primary rat Sertoli cell cultures PFNA reduced mRNA levelsof FSH-R, increased mRNA levels of ABP, and reduced mRNA levels of transferrin (Feng et al.
2010).
EDC KC4 Alters signal transduction in hormone- responsive cells factors and transcripts, andactivity
In a MVLN cell line, PFNA caused a significant downregulation in ER responsive genes (tff1
and egr3) in presence of E2 (Juan Li et al. 2020).
EDC KC6. Alters hormone synthesis
Gestational exposure to PFNA reduced testicular protein levels of StAR, and at 5 mg/kg-day
reduced testicular protein levels of SF1, CYP11A1, 3β-HSD, 17β-HSD and atered testiculargene expression of MIS was reported in mice (Singh and Singh 2019d). Increased brain
mRNA levels of the gene encoding LH was observed in male zebrafish (Zhang et al. 2016).
EDC KC8. Alters hormone distribution or circulating hormone levels
In addition to effects on levels of circulating reproductive hormones (discussed above inconjuction with Male KC8), alterations in other hormones reflective of EDCs were noted for
PFNA in human studies. These included decreased TSH levels in a cross-sectional study of
US subjects (Lewis et al. 2015). In animal studies, decreased TSH, total and free T4 in male

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SD rats (NTP 2019); increased T3 and T4 in zebrafish (Y Liu et al. 2011) and inhibition of T4TTR binding in a cell-free biochemical assay (Weiss et al. 2009) were observed.
EDC KC10. Alters fate of hormone-producing or hormone-responsive cells
Increased relative thyroid gland weight in male SD rats (NTP 2019), concentration-dependent
decrease in GH3 cell proliferation with PFNA in vitro (Long et al. 2013); and histologicalchanges in the thyroid follicles of F0 males in zebrafish (Y Liu et al. 2011) have been reported.

5. PFDA AND ITS SALTS: MALE REPRODUCTIVE TOXICITY
5.1 PFDA: Human studies of male reproductive effects
Eleven epidemiologic studies of possible effects of PFDA on the male reproductive systemwere identified. These studies examined one or more of the following types of outcomes:
anogenital distance (AGD); outcomes related to male reproductive function, including
reproductive hormones, semen quality parameters, and in vitro fertilization (IVF) outcomes;
and cancer. The studies are ordered by type of outcome, and within outcome type, by
publication date, in the following discussion and in Table 5.1. Some studies included multiple
outcomes but are included only once in the discussion and table. Statistically significant
results in Table 5.1 are in bold type. In the following summary, all results are statisticallysignificant unless otherwise noted.
Anogenital distance
The two prospective studies examining the association between early gestational PFDA
exposure and AGD in boys reported conflicting findings. The study by Tian et al. (2019) had alarger sample (n=500) and higher maternal PFDA concentrations (plasma median = 2.07, IQR= 1.34, 3.23 ng/mL), and reported an association with shorter anoscrotal distance (AGDAS) andanopenile distance (AGDAP) at birth. However, the differences were smaller and notstatistically significant at six months of age, and by 12 months, there were no longer
associations (Tian et al. 2019). In the study by Lind et al. (2017), the sample was smaller(n=299), and maternal serum PFDA concentrations were extremely low and had very little
variability: median = 0.3, IQR = 0.2, 0.39 ng/mL. Lind et al. reported that prenatal PFDA
exposure in the third quartile, compared to the first quartile, was associated with an increase in
AGDAS at three months, but no differences for AGDAP (Lind et al. 2017).

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Male reproductive function
Five cross-sectional studies examined associations between PFDA exposure and reproductive
hormones in adolescent or adult males. PFDA concentrations were low in all of these studies.
Zhou et al. (2016) reported that higher serum PFDA was associated with lower serum
testosterone (T) in a sample of 102 13- to 15-year-old boys. Other studies included adult maleparticipants and did not report consistent findings regarding reproductive hormones (Joensen
et al. 2009; Joensen et al. 2013; Ma et al. 2021; Petersen et al. 2018).
Four cross-sectional studies examined associations between PFDA and a variety of semenquality parameters, including indicators of sperm DNA integrity. The studies in China had thehighest PFDA concentrations and reported some associations with poorer semen quality. Pan
et al. (2019) had the largest sample (N=664) among the studies and measured both serum andsemen PFDA, and reported that higher semen PFDA was associated with decreased
progressive motility and straight-line velocity (VSL), and high DNA stainability (HDS) and DNA
fragmentation index (DFI), suggesting that PFDA exposure may be harmful to semen quality.
Serum PFDA was also associated with HDS (Pan et al. 2019). In a small cohort of couples
undergoing IVF due to female factor infertility, plasma PFDA was associated with decreased
sperm concentration and possibly reduced count and progressive motility (not statisticallysignificant), but not IVF outcomes (Ma et al. 2021). Other studies did not report results for
PFDA due to low exposure (Joensen et al. 2009), or reported few or no adverse effects.
CancerHardell et al. (2014) reported an association between PFDA and prostate cancer among men
with a first degree relative with prostate cancer. However, because PFDA was measured after
the cancer diagnosis, corroboration of this result is needed.
A case-control study by Lin et al. (2020) in Shanghai, China investigating the association
between serum PFDA levels and germ cell tumors in children did not report findings by tumor
location (e.g., testes vs. ovaries) or sex of the child (Lin et al. 2020). These limitations inreporting render the study non-informative for purposes of evaluating male reproductive
system hazards, and the study was not included in Table 5.1.

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Table 5.1 PFDA: Epidemiologic studies of male reproductive toxicity 4.
Reference; study
location, period,
design, sample
Lind et al., 2017
Denmark
2010-2012

Exposure matrix
andconcentration(ng/mL)
Maternal serum collected
at gestational weeks 5-12
(median 10 weeks)

Prospective cohort

Median (IQR) 0.3 (0.20.39) (from Table 2)

N=299

Quartiles

Odense Child Cohort: sonsof mothers who were >16
years old, of Western origin,
had no communication
barriers, delivered in areference hospital, and did
not use reproductive
assistance.

1st 0.1-0.19
2nd 0.2-0.29
3rd 0.3-0.39
4th 0.4-1.8

Outcomes
Anoscrotal distance(AGDAS), anopenile
distance (AGDAP), andpenile width at 3
months, measured threetimes by trained study
technicians

Results
AGDAS
β (95% CI) by PFDA quartile, mm:
1 Referent
2 0.6 (–1.1, 2.4)
3 2.1 (0.4, 3.9)
4 1.4 (–0.2, 3.1)
p-trend = 0.04
Continuous (with ln(PFDA)) 0.2 (–1.1, 1.6)
AGDAP

Covariates, other PFASs
analyzed
Adjusted for “post-conceptionalage” (sum of gestational (GA) atbirth and age of child at the AGD
measurement, in days), z-score forweight at 3 months, parity, maternalsmoking, and pre-pregnancy BMI
Other PFASs: PFHxS, PFOS,
PFNA

Comments
Correlations among PFASs
not reported.
Women who gave birth in2010 had lower PFDA
concentrations than women
who gave birth in 2011-2012.
Women who participated were
better educated, smoked less,and were more likely to be ofDanish origin than
nonparticipants.

β (95% CI) by PFDA quartile, mm:
1 Referent
2 0.5 (–1.5, 2.4)
3 1.9 (–0.1, 3.8)
4 –0.4 (–2.3, 1.5)p-trend = 0.97
Continuous (with ln(PFDA)) –0.6 (–2.0, 0.9)
Penile width: no association

Studies are ordered by outcomes (AGD, male reproductive function, prostate cancer), and within outcomes, by publication date. Statisticallysignificant results are in bold type.
4

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Reference; study
location, period,
design, sample
Tian et al., 2019
Shanghai, China
2012
Prospective cohort
N=500 male singletons born
to women who wererecruited at gestational
weeks 12-16 from one
hospital

Exposure matrix
andconcentration(ng/mL)

Outcomes

Maternal plasma (fasting)collected at gestational
weeks 12-16

AGD [AGDAS & AGDAP]
at birth, 6 months, and12 months

Median (5, 25, 75, 95
percentiles): 2.07 (0.72,
1.34, 3.23, 5.79)

Measured by trained
examiners with noknowledge of maternal
PFAS concentrations

Geometric mean (SD):
2.1 (1.92)

Resultsβ (95% CI) for unit change in ln(PFDA) (mm)Selected a priori and adjusted for:AGDAS

maternal age, education, parity,and pre-pregnancy BMI; GA atbirth, age at examination (days),infant size (weight at birth, weightfor-length z-score at 6 and 12
months of age)

Birth: –0.58 (–1.11, –0.06)
6 months: –1.14 (–2.93, 0.65)
12 months: no association
AGDAP
Birth: –0.63 (–1.24, –0.01)
6 months: –0.60 (–2.41, 1.20)
12 months: no association

Joensen et al., 2009

Serum

DenmarkMedian (5th, 95th

2003
Cross-sectional*
N=104
Healthy young men from thegeneral population reportingfor the military draft, medianage 19 years.

percentiles)

All 0.9 (0.3, 1.1)
High T 0.9 (0.2, 1.1)
Low T 0.8 (0.4, 1.2)

Serum reproductive
hormones and related
proteins: T, E2, SHBG,
LH, follicle-stimulating
hormone (FSH), inhibin
B, free androgen index(FAI)

Covariates, other PFASs
analyzed

No associations with T
Due to low concentration, PFDA was not includedin further analyses

Stratified by breastfeeding duration
for AGD at 6 and 12 months
8 PFASs detected in ≥80% ofparticipants: PFHxS, PFOA, PFOS,
PFNA, PFDA, PFUnDA, PFDoA,
PFTrDA

Comments
Pearson correlation coefficient
for ln-transformed PFDA andPFOS r = 0.73
PFOA r = 0.48
PFNA r = 0.79
PFUnDA r = 0.86
PFDoA r = 0.57
PFTrDA r = 0.33
75% of mothers had
university- level education or
higherPFDA was included only inbivariate analyses (no adjustments)
Other PFASs: PFHxS, PFPA,PFOA, PFOS, PFOSA, PFNA,PFUnDa, PFDoA, PFTrA

Semen quality: semen
volume; sperm
concentration, count, %motile, morphology*Men were selected forhighest and lowest T levels:
Group 1, n=53, median 31.8
nmol/LGroup 2, n=52, median 14.0
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Reference; study
location, period,
design, sample

Exposure matrix
andconcentration(ng/mL)

Joensen et al., 2013

Serum

Denmark

Mean ± SD 0.38±0.16

2008-2009Median (IQR) 0.35 (0.28,
0.45)

Cross-sectional
N=247
Healthy young men
randomly selected from thegeneral population, medianage 19 years

Outcomes
Serum reproductive
hormones and related
proteins: total T, free T,
E2, inhibin-B, FAI;
T×100/SHBG), FSH,
LH, SHBG
Semen quality: volume;
sperm count,concentration, motility,
morphology, total
normal sperm countTesticular volume

Results
Hormones (all ln-transformed)
NS association with lower: T, FAI, free T, free
T/LH, T/LH, E2, inhibin-B/FSH
NS association with ↑ FSH
No association with T/E2, SHBG, LH, inhibin-B,
FAI/ LH

Covariates, other PFASs
analyzed

Comments

Analyses with hormones and SHBG
were adjusted for BMI andsmoking.

Small range of PFDA
concentrations in study
participants

Semen volume, concentration, andtotal count were adjusted for
abstinence time.

Participation rate was ~30%,
which is “higher than otherpopulation-based semenquality studies”.

Semen quality% morphologically normal sperm
was unadjusted.

No associations with semen volume,
concentration, total count, % morphologicallynormal, total normal count.

Progressively motile % wasadjusted for time to semen
analysis.

NS association with ↓ % progressively motile
sperm β=-1343 (-2759, 73.692) (adjusted for timeto semen analysis)

Considered but not included: timeof day of blood sample, ethnicity,
recent alcohol use, prenatal
exposure to tobacco smoke,previous or current diseases, recent
fever, recent medication use,season, interaction with cigarettesmoking.

Testicular volume
No association

Participants were recruited at
a compulsory medical
examination for consideration
for military service
Men were asked to abstain for
48 hours; median abstinence
period was 62 hours.
Analyses were blinded

Other PFASs: PFHxS, PFHpS,PFOA, PFOS, PFNA

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Reference; study
location, period,
design, sample

Exposure matrix
andconcentration(ng/mL)

Outcomes

Results

Louis et al., 2015

Serum

Semen quality:

Associations with 1-unit increase in ln(PFDA):

Michigan and Texas

Median (IQR); geometricmean (95% CI)

Volume, straw distance,
sperm concentration,
total count, hypoosmotic swollen,

No significant differences in semen volume;
sperm viability, count, or concentration.

2005-2009
Cross-sectional
N=501
Population-based sample ofcouples planning pregnancy,
recruited through amarketing database in
Michigan and afishing/hunting license
registry in Texas.
Zhou et al., 2016
Taipei, Taiwan
2009-2010
Cross-sectional

Michigan 0.3 (0.2, 0.4);
0.31 (0.28, 0.35)
Texas 0.5 (0.3, 0.6); 0.47
(0.45, 0.50)

8 motility measures,
6 sperm headmeasures,12 individual and 2
summary morphology
measures,

Sperm head length, β=–0.155 (–0.304, –0.006)µm% of sperm with coiled tail, β=–7.603 (–14.014,–1.193)

Covariates, other PFASs
analyzed
Adjusted for age, BMI, serum
cotinine, abstinence, sample age,research site (Texas/Michigan)
Other PFASs: PFOA, PFOS,PFOSA, Et-PFOSA-AcOH, MePFOSA-AcOH, PFNA

No significant associations with other semen
parameters

Median (IQR) 0.9 (0.8,
1.1)

Serum T and E2(average of 2 values)β (95% CI) per ng/mL increase in PFDA:
Ln(T) –0.2565 (–0.4135, –0.0994) mmol/L
Ln(E2) 0.0734 (–0.1189, 0.2657) pmol/L

Semen provided after 2-day
abstinence. A 2nd sample
collected approximately 1month later was used tocorroborate azoospermia
observed in the 1st sample.
Semen analysis was
performed one day aftercollection.
PFOSA, Me-PFOSA-AcOH,
and PFOA were most often
associated with semen
parameters.

2 sperm chromatinstability measures
Serum sampled after 8hour fast

Comments

Age, parental education, BMI,
environmental tobacco smoke
exposure, regular exercise, monthof survey

More associations between
individual PFAS levels andhormone levels were reported
in boys than in girls

Other PFASs: PFBS, PFHxA,
PFHxS, PFOA, PFOS, PFNA,
PFDoA, PFTA

N=102 healthy 13-15-yearold boys from 7 publicschools
From the control cohort of
the Genetics andBiomarkers study forChildhood Asthma

PFNA and its salts andPFDA and its salts

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Reference; study
location, period,
design, sample

Exposure matrix
andconcentration(ng/mL)Petersen et al., 2018

Serum

Faroe Islands

Median (Range) 0.52
(0.16-4.59)

2007-2009
Cross-sectional
N=263
Entire population of 24-26
year old men were invited

Pan et al., 2019
Nanjing, China
2015-2016
Cross-sectional
N=664 males from couples
visiting a reproductive
medical center. Some men
had fecundity issues andsome were partners of
women with female factor
infertility.

Outcomes
Serum reproductive
hormones and related
proteins: FSH, inhibin-B,
LH, T, FT, estradiol,
SHBG, Inhibin-B/FSH,
T/LH, FT/LH,
T/estradiol, FT/estradiol)

Results
Specific results for PFDA were not reported.
Authors state there were no associations
between semen quality and any PFASs.
Associations with reproductive hormones were
not mentioned for PFDA.

Semen quality: Spermconc., total sperm count,semen volume, % motile
sperm
Semen (after 2-day
abstinence):
Median (IQR) 0.020(0.012, 0.034)

Semen quality:
semen volume,
sperm conc.,

Serum (same day as
semen)

sperm count,

Median (IQR) 1.240(0.706, 2.031)

curvilinear velocity(VCL),

Exclusions: severe
reproductive tract diseaseprogressively motile (%),

straight-line velocity(VSL),
morphologically normal(%),
DNA fragmentationindex (DFI),
high DNA stainability(HDS)

Covariates, other PFASs
analyzed
Reproductive hormones (except
FSH, LH) were adjusted for BMI,
smoking, age, time of day of blood
sampling.

Comments
49% of those reached byphone (24% of all young men
invited by mail) participated.

Sperm conc., total sperm count,and semen volume were adjusted
for abstinence (hours).
% motile sperm was adjusted forinterval between ejaculation andassessment.
Other PFASs: PFHxS, PFOA,PFOS, PFNA

P-values were adjusted for false discovery
rate/multiple comparisons
Change (95% CI) per unit increase in:ln(semen PFDA)
Progressively motile (%) β=–1.475 (–2.796, –0.155) p=0.1
VSL (µm/s) β=–0.659 (–1.305, –0.013) p=0.1
DFI (%) β=0.083 (0.021, 0.145) p=0.05
HDS (%) β=0.068 (0.026, 0.110) p=0.02
No associations with other semen parameters
Ln(serum PFDA)HDS (%) β=0.073 (0.031, 0.115), p=0.02

Adjusted for: age, BMI, BMI2,
smoking status, alcohol use,abstinence time
Also considered: having fathered apregnancy, occupational hazards,
and medical history
Analyses of associations withsemen parameters focused on themost abundant PFASs in serumand semen and those detected in atleast 80% of semen samples:
PFOA, PFOS, PFNA, PFUnDA, 6:2
Cl-PFESA.

Correlation between serumand semen PFDA: r=0.74
PFDA correlations with other
PFASs:PFHxS r=0.327
PFOA r=0.428
PFOS r=0.688
PFNA r=0.825
PFUnDA r=0.851
Associations of PFDA withsemen parameters weresimilar to those of other
PFASs.

No associations with other semen parameters

Concentration andmotility were measured
by CASAPFNA and its salts andPFDA and its salts

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Reference; study
location, period,
design, sample

Exposure matrix
andconcentration(ng/mL)

Ma et al., 2021

PlasmaHangzhou, Zhejiang, ChinaMedian (IQR) [range]2017

2.1 (1.4, 4.2) [0.5, 19.5]

Prospective cohort (in vitro
fertiliization (IVF) outcomes)
and cross-sectional(hormones and semenquality)Concentrations by tertiles:

N=96 couples undergoing
IVF treatment due to tubal
factor infertility

1st: 0.5-1.6
2nd: 1.6-3.6
3rd: 3.7-19.5

Outcomes
Plasma reproductive
hormones: FSH, LH, E2,T

Results
Compared to 1st tertile of PFDA:
Hormones

Semen quality:

No associations with FSH, LH, or E2.

volume; sperm count,concentration, motility,
morphology

Semen quality

IVF outcomes

NS association with ↑ T in 2nd and 3rd tertiles, ptrend=0.05

Covariates, other PFASs
analyzed
Adjusted for age, BMI, smoking
statusOther PFASs: PFBA, PFHxS,
PFHpA, PFOA, PFOS, PFOSA,PFNA, PFUnDA, PFDoA

Each couple contributed one
cycle of IVF treatment to thestudy.
PFDA was strongly correlated
with:
PFOS r = 0.722
PFNA r = 0.881

Sperm concentration ↓ in 2nd (ns) and 3rd
tertiles (24% reduction), p-trend = 0.072

PFUnDA r = 0.943
PFDoA r = 0.856

Sperm count ns ↓ in 3rd tertile, p-trend=0.111

2nd and 3rd tertile PFUnDA
was associated with ↑ T% sperm with normal morphology ns ↓ in 2nd and3rd tertiles, p-trend=0.150

Men with severe male-factor
infertility were excluded

Comments

Progressive motility no association
IVF outcomes
No association with fertilization, number of goodquality embryos at day 3, implantation, clinicalpregnancy, or live births.

Hardell et al., 2014
Sweden, 2007-2011
Case-control

Whole blood drawn after
diagnosis but before
treatment with radiation or
chemotherapy.

N=200 cases of newly
diagnosed prostate cancerfrom one hospital

Mean, median (range)

N=181 population-based,
matched controls with nohistory of cancer

Controls 0.291, 0.269(0.0244, 1.0)

Cases 0.338, 0.301(0.0300, 1.2)

PFNA and its salts andPFDA and its salts

Prostate cancer
Medical records

OR = 1.4, 95% CI (0.9, 2.1) for exposure above
control median

Adjusted for age, BMI, year ofsample.

Apparent gene / environment
interaction.

Among men with 1st degree relative withprostate cancer:

Matched on age and geographic
area.

OR = 2.6 (1.1 - 6.1) for exposure above control
medianOther PFASs: PFHxA, PFHxS,PFOA, PFOS, PFNA, PFUnDA,
PFDoDA

Participation rates were 60%
for controls and 79% forcases.

Using 75th percentile exposure cutoff did notchange results.

Gleason score, prostatespecific antigen, and a
combination of both were notassociated with PFASs.
Because serum was sampled
after cancer diagnosis, thepossibility of reverse
causation cannot be excluded

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5.2 PFDA: Animal studies of male reproductive toxicity
Several in vivo animal studies investigating the effect of PFDA on outcomes relevant tomale reproductive toxicity (e.g., effects on the gonads, reproductive hormone levels)
were identified, including multiple studies in male rats (Bookstaff et al. 1990; George
and Andersen 1986; NTP 2019; Olson and Andersen 1983; Van Rafelghem et al.
1987b), one study in male mice (Van Rafelghem et al. 1987b), one study in male
hamsters (Van Rafelghem et al. 1987b), one study in male guinea pigs (Van Rafelghem
et al. 1987b), and one study in zebrafish (Jo et al. 2014) (See Table 5.2.2). Animalstudies that administered PFDA as part of a mixture of chemicals were not included inthis review.
Of the multiple studies of PFDA conducted in male rats, one study treated male SD rats
at five dose levels (0.156-2.5 mg/kg-day) by daily gavage for 28 days (NTP 2019). Allother rat studies assessed the effects of PFDA seven to 30 days after administration ofa single intraperitoneal (i.p.) injection of relatively high doses (ranging from 20-80
mg/kg). The studies in male mice, hamsters, and guinea pigs also treated the animals
with a single i.p. injection of relatively high doses of PFDA and evaluated the toxic
effects 14 to 28 days after dosings (Van Rafelghem et al. 1987b). In addition to thesestudies in rodents, the effects of 120-day exposure to PFDA on sex steroid hormonesand expression of mRNA of selected genes in the hypothalamic-pituitary-gonadal-liver(HPGL) axis were evaluated in zebrafish (Jo et al. 2014).
Similar to the studies on PFNA (see Section 4.2), all the studies in rats, mice, hamsters,
or guinea pigs used relatively short period of treatment and/or observation. None of thestudies in rats or mice treated or observed the animals for one spermatogenic cycle,
which is approximately 35 days in mice and 54 days in rats (Clermont 1972; Cordeiro etal. 2021; Johnson et al. 2000). The period of treatment or observation is also shorter
than the half-life (>40 days) of PFDA in rats or mice (Kudo et al. 2001; Ohmori et al.
2003). Should the treatment or observation period last more than one spermatogenic
cycle, histopathological changes could become more obvious and/or observed at lower
doses.
Major findings from these studies are discussed below, grouped by type of effect(reproductive organ weight and histopathology, sperm parameters, hormonal effects,
and effects on fertility).
Organ weights & histopathology
PFDA-induced histopathological changes in the testis have been observed in thestudies conducted in rats, hamsters, and guinea pigs, but not in the one study
conducted in mice, which examined the testes 28 days after a single i.p. injection. In
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the NTP 28-day study in SD rats aged 10-14 weeks at the beginning of treatment,
PFDA at doses ≥1.25 mg/kg-day reduced epididymal weight by 10-23% and interstitialcell atrophy in 8-10 of 10 rats in each group (NTP 2019). At the high dose of 2.5 mg/kgday, spermatid retention in seminiferous tubules (inhibited spermiation) was observed in
4 of 10 animals. Minimal germinal epithelium degeneration in the testes and exfoliatedgerm cells in epididymal ducts were also observed in rats treated with 2.5 mg/kg-day
PFDA. Design parameters and key findings from this NTP study are presented in Table
5.2.1.

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Table 5.2.1. PFDA or Wyeth-14643: Relevant key findings in male rats treated by gavage for 28 days (NTP 2019).
Treatment

Dose (mg/kg-day)
PFDA

Wyeth-14643

0

0.156

0.312

0.625

1.25

2.5

25 #

10

10

10

10

10

10

10

333±6

344±7

331±6

322±5

263±4**

207±8**

308±5**

Relative liver weight (g)35.50±0.9739.32±0.53**

42.61±0.56**

45.56±0.84**

54.77±0.68**

67.90±1.19**

53.90±1.67**

Hepatocyte cytoplasmic
alteration

0/10

0/10

0/10

8/10**

10/10**

10/10**

10/10**

Hepatocyte hypertrophy

0/10

0/10

0/10

0/10

8/10**

10/10**

10/10**

Relative thyroid weight(g)

0.07±0.00

0.07±0.00

0.07±0.00

0.08±0.00

0.10±0.00**

0.10±0.00**

No data

TSH (ng/dL)

19.79±3.75

16.16±1.88

17.73±2.70

15.52±2.50

11.63±1.18

8.97±2.28

13.07±1.01**

T3 (ng/dL)

95.86±4.01

72.87±2.66

66.14±2.8474.74±5.45147.95±9.69

179.99±13.73

No data

Free T4 (ng/dL)

2.02±0.21

1.90±0.22

1.17±0.11**

1.13±0.06**

0.65±0.05**

0.36±0.05**

1.31±0.13**

Total T4 (ng/dL)

4.36±0.32

4.27±0.23

3.24±0.18*

3.82±0.09

4.59±0.26

4.64±0.15

2.78±0.17**

Absolute testis weight (g)1.786±0.06

No data

No data

1.777±0.031

1.730±0.031

1.594±0.047**

1.774±0.036

Absolute epididymal
weight (g)0.528±0.015

No data

No data

0.508±0.009

0.474±0.013*

0.407±0.024**

0.512±0.011

Spermatid heads(106/testis)230.3±12.5

No data

No data

208.2±8.8181.6±10.7*

217.0±7.5255.8±11.5

Epididymal sperm count(106/cauda epididymis)

136.3±10.2

No data

No data

120.8±5.5112.9±7.3

95.7±11.5**

116.3±8.4

Epididymal sperm motility

85.7±0.7

No data

No data85.5±1.0

84.1±0.776.2±7.888.2±0.5

T (ng/mL)

2.59±0.65

6.36±1.69

3.42±0.87

1.94±0.41

0.93±0.19*

0.65±0.26**

1.56±0.39*

No. of rats per group
Body weight (g)PFNA and its salts andPFDA and its salts

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Treatment

Dose (mg/kg-day)
PFDA

Wyeth-14643

0

0.156

0.312

0.625

1.25

2.5

25 #

Interstitial cell atrophy

0/10

0/10

0/10

0/10

8/10**

10/10**No data

Spermatid retention

0/10

0/10

0/10

0/10

0/10

4/10*

No dataGerm cell degeneration

1/10

0/10

0/10

0/10

0/10

4/10

1/10

Epididymal lesion

1/10

0/10

0/10

0/10

0/10

4/10

1/10

# Only the findings from the high dose of Wyeth presented in this table. *statistically significant, p<0.05. **statisticallysignificant, p<0.01.

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In addition to the NTP study in SD rats, degeneration in testicular tissues was observedin male Fischer rats 8 or 16 days after a single i.p. injection of 50 mg/kg of PFDA(George and Andersen 1986; Olson and Andersen 1983; Van Rafelghem et al. 1987b).
Bookstaff et al. found no histopathological changes in the testis of adult SD rats sevendays after a single i.p. injection of 20, 40 or 80 mg/kg PFDA (Bookstaff et al. 1990).
However, the authors found that PFDA at 40 or 80 mg/kg caused significant reduction inplasma levels of T and DHT as well as atrophy of seminal vesicles and prostates. The
testis weight was also significantly reduced after PFDA i.p. injection at 80 mg/kg.
In the study comparing the liver and testicular effects of PFDA among rats, mice,
hamsters, and guinea pigs, Van Rafelghem et al. treated the animals with a single i.p.
injection of PFDA. (Van Rafelghem et al. 1987b). Necropsy was performed on all
surviving animals on Day 14 (guinea pigs), Day 16 (rats and hamsters), or Day 28(mice) after dosing. The authors found degenerative changes in the testis of hamsters(Day 16 at 50 or 100 mg/kg) and guinea pigs (Day 14 at 150 mg/kg) were not as severe
as in rats. Single i.p. injections of PFDA at doses up to 250 mg/kg did not cause obvious
histopathological lesions in the testis of treated mice on Day 28 after dosing.
Several of the studies discussed above found that PFDA at the dosing levels toxic to rat
testes also caused reduced food intake and body weight, liver damage, or other toxic
effects. As cited in the 2019 NTP study report, Chapin and Creasy had reported that
decreased body weight gain and/or decreased food intake may result in decreased T
production through decreased GnRH release and cautioned that such effects should not
be mistaken for a direct effect of the test material on androgen balance (Chapin andCreasy 2012). Studies investigating the effects of severe food restriction in rats for as
long as 17 weeks that resulted in reductions in body weight of up to 30% did notobserve effects on fertility, testicular weight, or severe histopathological changes(Chapin et al. 1993; Rehm et al. 2008). Therefore, PFDA-induced testicular effects areunlikely to be secondary to the general toxicity observed in the same animals.
Sperm parameters
Reduced epididymal sperm count was observed in SD rats following 28-day oral
treatment with PFDA at 2.5 mg/kg-day (NTP 2019). There is no other whole animal
study that assessed sperm parameters following exposure to PFDA.
Hormonal effects
PFDA-produced reduction in serum or plasma level of T was observed in rats following
28-day oral treatment at ≥1.25 mg/kg-day doses (NTP 2019) or after a single i.p.
injection at 40 or 80 mg/kg (Bookstaff et al. 1990). Plasma DHT levels were also
decreased by PFDA in the studies by Bookstaff et al. 1990, while no treatment-related
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effect was observed on plasma LH. Bookstaff et al. showed PFDA had no effect on
plasma T levels in castrated rats supplemented with normal levels of systemic
testosterone via testosterone implants, but that PFDA treatment of intact rats decreased
the dose-dependent secretion of T in response to human chorionic gonadotropin (hCG)
stimulation in studies with decapsulated testes, Based on these studies, Bookstaff et al.
suggested that PFDA reduces plasma T in vivo by disrupting testicular response to LH
stimulation (Bookstaff et al. 1990).
The effects of PFDA on steroidogenesis were also evaluated in zebrafish (Jo et al.
2014). In this study, freshly fertilized zebrafish eggs were collected and exposed to
PFDA at concentrations of 0, 0.01, 0.1 and 1.0 ml/L for 120 days. Compared to controls,
the ratios of E2 to T and E2 to 11-ketotestosterone (11-KT) were significantly reduced in
the plasma of zebrafish exposed to 1.0 m/L PFDA. Exposure to PFDA at 1.0 mg/L also
up regulated transcription levels of the cyp19a gene in male gonads. The authors
concluded that PFDA caused estrogenic effects in male zebrafish.
Effects on fertility
There is no study available on the effects of PFDA on male-mediated fertility.

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Table 5.2.1 PFDA: Evidence on the male reproductive toxicity in animal studies.
Study Design
NTP 2019
Male SD rats, 10-11 weeks old at the
beginning of dosing, 10 rats per dosegroup.
Treatment: PFDA (>97% purity, i.e., linear
PFDA), once daily by gavage in deionizedwater with 20% Tween® 80 at doses of 0,0.156, 0.312, 0.625, 1.25, or 2.5 mg/kgday, respectively, for 28 days. Six other
PFASs and Wyeth-14,643 (a PPARαagonist) were also tested for comparison.

Olson and Andersen 1983
Male Fischer rats, weighing 200 g. A totalof 24 rats per group. Six animals pergroup were sacrificed on days 2, 4, 8 and
16 after treatment.
Treatment: single i.p. injection of 50
mg/kg PFDA (purity not reported) inpropylene glycol-water (1:1) solution, andtwo control groups: vehicle controls andpair-fed vehicle controls.

PFNA and its salts andPFDA and its salts

Outcomes Assessed
General toxicity: survival, bodyweights, clinical chemistry,
thyroid hormones, liver effects(e.g., gene expression, enzyme
activity) and histopathology.
Male reproductive toxicity: testis
and epididymis weights; sperm
production and motility;
histopathology of the testis andepididymis; serum T.

General toxicity: survival, foodintake, body weight, organ
weights, fatty acid level in liverand blood, and general
histopathology.
Male reproductive toxicity: testis
weightMajor Findings
General toxicity:
No effect on survival; lower body weights at ≥ 1.25 mg/-kgday; reduced free T4 concentrations at ≥ 0.312 mg/kg-day;
Increased relative weights of liver, kidney, and adrenal
gland; increased hepatic expression of PPARα -associatedgenes (Acox1, Cyp4a1) and constitutive androstane
receptor (CAR)-associated genes (Cyp2b1, Cyp2b2) at ≥0.156 mg/kg-day.
Male reproductive toxicity:
Dose-dependent trends in reduction in testis and epididymis
weights, serum T level and epididymal sperm count, withstatistically significant differences (p < 0.05) at doses of≥1.25 mg/kg-day (T level and epididymal weights) or 2.5
mg/kg-day (testis weight, epididymal sperm count).
Interstitial cell atrophy in 8/10 and 10/10 rats at 1.25 and 2.5
mg/kg-day doses, respectively. At 2.5 mg/kg-day, 4/10
animals had germ cell degeneration, inhibited spermiation,and epididymal lesions.
General toxicity: Deaths occurred in the 2nd and 3rd weeks
after dosing with PFDA. Severely reduced food consumption
and body weight, beginning one day after dosing, andcontinuing to study termination. Significantly increased liver
weight on days 8 and 16 compared to pair-fed controls;
decreased liver weight on days 8 and 16 compared to vehicle
controls.
Male reproductive toxicity: Significantly decreased testis
weight on day 16 compared to vehicle and pair-fed controls(1.7±0.1 g vs 2.8±0.1 g in vehicle controls and 2.2±0.0 g inpair-fed controls).

88

Notes
Treatment period of 28days is shorter than one fullcycle of spermatogenesis inrats (about 56 days).
Similar effects wereobserved with PFNA
treatment.
Treatment with Wyeth14,643 caused significant
effects in the liver, but notin the testis, indicating
testicular effects of PFDA
may not be mediated via
the PPARα pathway.

The dose used in this study
is higher than the acute 30day LD50 following i.p.
injection (41 mg/kg).
The authors noted findings
from an earlier study,
published only as an
abstract, in which a single
dose of 100 mg/kg PFDA tomale Fischer rats resulted
in seminiferous tubule
atrophy by day 8 and franknecrosis and calcification
by day 16.

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Study Design

Outcomes Assessed

Major Findings

Notes

George and Andersen 1986

General toxicity: food intake,body weight, organ weights,
clinical chemistry, fatty acidlevel in liver and whole blood,and general histopathology.

General toxicity: Severely reduced food consumption andbody weight, beginning one day after dosing. Compared topair-fed controls body weights were significantly decreased,
and liver weights were significantly increased. Increase in
several fatty acids in liver and in thymic atrophy.

Survival not reported. The
dose used in this study ishigher than the acute 30day LD50 following i.p.
injection (41 mg/kg).

Male reproductive toxicity:histopathology of the testis

Male reproductive toxicity: Atrophy and degeneration of theseminiferous tubules were first seen at day 16 in treated rats
and remained severe at day 30.

General toxicity: mortality, foodintake, body weight, liver weight

General toxicity: increased mortality by day 16: 0/5 and 1/5
in control and 50 mg/kg dose groups, respectively. Rapid
and immediate reduced food intake and body weight.
Increased relative liver weight.

Male Fischer 344 rats, weighing 225-250
g. At least six rats per group weresacrificed on days 4, 8, 12, 16 or 30 after
treatment.
Treatment: PFDA (96% purity, i.e., linear
PFDA) in propylene glycol- water (1:1)
solution, single i.p. injection of 50 mg/kg,
and pair-fed vehicle controls.
Van Rafelghem et al. 1987b
Male Fischer 344 rats weighing 200-250
g, five rats per group (this study compared
the toxic effects of PFDA in rats, mice,
hamsters, and guinea pigs).

Histologic changes in rat
liver, thymus, and testes
have been described inprevious studies:
Liver - heptomegaly andswelling of liver
parenchymal cells
characterisitic ofperoxisome proliferation

Treatment: single i.p. injection of 50
mg/kg PFDA (purity not reported) inpropylene-water (1:1) solution, andvehicle controls. All animals weresacrificed on day 16.

Thymus - thymic atrophy
Testes – testicular atrophy.

Van Rafelghem et al. 1987b
Male Syrian golden hamsters weighing
100-120 g, four hamsters per group.
Treatment: single i.p. injection of 50, 100,
200 or 400 mg/kg PFDA (purity notreported) in propylene-water (1:1)
solution, and vehicle controls. All animals
were sacrificed on day 16.

PFNA and its salts andPFDA and its salts

General toxicity: mortality, foodintake, body weight, liver
weight, histopathology of liver
and thymus.
Male reproductive toxicity:histopathology of the testis

General toxicity: dose-related increase in mortality by day
16: 0/4, 0/4, 2/4, 4/4, and 4/4 in control, 50, 100, 200, and
400 mg/kg dose groups, respectively. Rapid and immediate
reduced food intake and body weight. Increased relative
liver weight at 50 mg/kg (LD50), heptomegaly and swellingof parenchymal cells characterisitic of peroxisome
proliferation, and thymic atrophy.
Male reproductive toxicity: mild seminiferous tubular
degeneration with decreased spermatogenesis in surviving
hamsters at 100 mg/kg, in 1/4 animals at 50 mg/kg and 0/4
in vehicle controls. Decrease testis weight in treatedhamsters (data not shown).

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Study Design
Van Rafelghem et al. 1987b
Male CF-1 mice weighing 30-40 g, ten
mice per group.
Treatment: single i.p. injection of 150,
200, or 250 mg/kg PFDA (purity notreported) in propylene-water (1:1)
solution, and vehicle controls. All animals
were sacrificed on day 28.
Van Rafelghem et al. 1987b
Male Hartley guinea pigs weighing 535660 g, three animals per group.
Treatment: single i.p. injection of 125,150,
or 175 mg/kg PFDA (purity not reported)in propylene-water (1:1) solution, andvehicle controls. All animals weresacrificed on day 14.

PFNA and its salts andPFDA and its salts

Outcomes Assessed

Major Findings

General toxicity: mortality, bodyweight, liver weight,
histopathology of liver andthymus.

General toxicity: dose-related increase in mortality by day
28: 0/10, 6/10, 10/10, and 10/10 in control, 150, 200, and
250 mg/kg dose groups, respectively. Rapid and immediate
reduced body weight. Increased relative weight at 150
mg/kg (LD50), heptomegaly and swelling of parenchymal
cells, characterisitic of peroxisome proliferation. No
histologic effects on the thymus.

Male reproductive toxicity:histopathology of the testis

NotesMale reproductive toxicity: no histologic effects on the testes
at day 28 in any dose group. A slight decrease in testes
weight in treated mice was reported (data not shown).
General toxicity: mortality, bodyweight, liver weight,
histopathology of liver andthymus.
Male reproductive toxicity:histopathology of the testis

General toxicity: increased mortality by day 14: 0/3, 1/3, 0/3,
and 2/3 in control, 125, 150, and 175 mg/kg dose groups,
respectively. Rapid and immediate reduced body weight.
Mild to moderate periportal hepatocellular cytoplasmic
alterations at 150 and 175 mg/kg. No clear histologic effects
on the thymus.
Male reproductive toxicity: no degenerative changes in thetestes of animals at 125 mg/kg; mild to moderate
degeneration in the seminiferous epithelia at 150 mg/kg; noeffects observed in the testes of the one surviving guinea pig
at 175 mg/kg. Decreased testes weight in treated guinea
pigs (data not shown).

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Study Design
Bookstaff et al. 1990
Adult male SD rats weighing
approximately 295 g. Ten rats per group.
Treatment: PFDA (96% purity) inpropylene glycol/water (1:1) solution, asingle i.p injection of 20, 40 or 80 mg/kg
and two control groups: vehicle controls
and pair-fed vehicle controls. All animals
were sacrificed on day 7 after dosing.

Outcomes Assessed

Major Findings

Notes

General toxicity: feed
consumption and body weight.

General toxicity: reduced feed consumption and body weightat 80 mg/kg compared to vehicle controls, and reduced bodyweight at 40 mg/kg compared to vehicle and pair-fed
controls (on day 7 post-dosing).

Depending on the germ cell
population targetted by
PFDA, testicular lesions may
not be obvious on day 7
after a single injection.

Male reproductive toxicity:
Plasma levels of T, DHT and
LH; histology and organ weightsof the testis, seminal vesicles
and ventral prostate.

Experimental Model: in vitro incubation of
decapsulated testis from adult rats 7 days
after a single i.p. injection of 0, 20, 40, or
80 mg/kg PFDA (96% purity).
Treatment: decapsulated testis was
incubated in the absence or presence of
100 mIU human chorionic gonadotropin(hCG)/mlfor 3 hours.

PFNA and its salts andPFDA and its salts

Reduction in plasma levels of T and DHT at 40 and 80
mg/kg compared to vehicle and pair-fed controls. No effecton plasma concentrations of LH.
Reduction in the weight of testis at 80 mg/kg compared tovehicle control, and in the weights of seminal vesicles andventral prostate at 20, 40 and 80 mg/kg compared to vehicle
and pair-fed controls (seminal vesicle weight at 20 mg/kg
was decreased compared to vehicle control only). Apparent
atrophy of the epithelium of seminal vesicles at 80 mg/kg,
and of the ventral prostate at 40 and 80 mg/kg. No
histological changes in the testis.

In a parallel experiment, rats (6 per group)
were castrated and implanted withsustained-release testosterone-containing
capsules two hours after receiving asingle i.p injection of PFDA at 20, 40 or 80
mg/kg, or vehicle (vehicle controls andpair-fed controls). Animals were sacrified
on day 7 after dosingand the testes wereused in the in vitro experiments(summarized below).
Bookstaff et al. 1990

Male reproductive toxicity:

The authors concluded thatPFDA disrupted thefeedback relationshipbetween plasma androgenand LH concentrations intreated animals.

In castrated/testosterone-implanted rats, seminal vesicle
weight was decreased at 20 and 40 mg/kg compared to pairfed controls. PFDA treatment had no effect on ventral
prostate weight or plasma T levels.
hCG-stimulated T secretion,measured as the difference
between basal T secretion (inthe absence of hCG) and hCGstimulated secretion over a 3hour period

Dose-related decrease in hCG-stimulated T secretion by
decapsulated testes from rats treated with PFDA at 40 and
80 mg/kg, compared to testes from vehicle and pair-fed
controls.

91

The authors concluded thatPFDA decreases testicularresponsiveness to LH-like(hCG) stimulation.

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Study Design
Jo et al. 2014
Zebrafish, four replicates (with 10
fertilized eggs each) per group. Threemale fish were randomly sampled fromeach of four replicate tanks per group forhormone and gene expression
measurements
Treatment: PFDA (98% purity) atconcentrations of 0, 0.01, 0.1, 1.0 or 10
mg/L in water from 1 day post fertilization(dpf) to 120 dpf.

Outcomes Assessed
General toxicity: survival at 17
dpfae), 34 and 61 dpf (juvenile),and 120 dpf (adult); length, wetweight, and condition factor.
Male reproductive toxicity: bloodlevels of E2, T, and 11-KT.
mRNA expression of genes inthe HPGL axis

Major Findings
General toxicity:
Survival at 17 dpf was significantly decreased atconcentrations of 0.1 mg/L and above, compared tocontrols. The no observed effective concentration was 0.01
mg/L. 100% mortality occurred in the 10 mg/L group by 34
dpf. Treatment had no effect on length, weight or condition
factor.
Brain: increased mRNA expression of cyp19b, erα, and er2β
at 1 mg/L.
Gonad: increased mRNA expression of cyp19a at 1 mg/L.
Liver: increased mRNA expression of vtg1 at 1 mg/L.

Notes
The authors noted that
increased ratios of E2/T
and E2/11-KT and
increased gene expressionof vtg1 suggest that PFDA
has estrogenic effects inzebrafish.
In an in vitro study reported
in the same paper, PFDA
had no effect on E2, T, or
E2/T in H295R cells, ahuman adrenocorticalcarcinoma cell line.

Male reproductive toxicity:
No effect on blood levels of E2, T, or 11-KT, compared tocontrols. Increased ratio of E2/T and E2/11-KT at 1.0 mg/L,
compared to controls.

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5.3 PFDA: Mechanistic considerations and other relevant data
The mechanistic literature regarding PFDA and male reproductive toxicity is sparse. We
provide a brief overview of findings from general toxicity studies of PFDA and studies ofgeneral mechanisms of PFDA-induced cytotoxicity, followed by discussion of studies
that may be more informative with respect to the effects PFDA may have on the male
reproductive system.
General toxicity
In male Sprague-Dawley rats, a single i.p. dose of PFDA resulted in a LD50 of 41 mg/kg(30-days), a LD50 of 64 mg/kg (14 days), and at 50 mg/kg PFDA caused significant
reduction in food intake, decreased body weight, and mortality (Olson and Andersen1983). Other studies have observed similar effects of PFDA on food intake, bodyweight, and mortality. For example, a study of male rats treated with a single dose ofeither 40 mg/kg or 80 mg/kg PFDA reported reduced body weight at both doses, as well
as reduced food consumption at the higher dose (Bookstaff et al. 1990). Some authors
suggested these effects of PFDA are similar to the wasting syndrome observed in rats
exposed to TCDD (George and Andersen 1986; Olson and Andersen 1983; Van
Rafelghem et al. 1987a; Van Rafelghem et al. 1987b). In other studies, conducted infemale Sprague-Dawley rats, Borges et al. 1993 found that PFDA is a noncompetitiveand reversible inhibitor of the peroxisomal enzymes fatty acyl-CoA oxidase (HYDRA)
and 3-hydroxyacyl-CoA dehydrogenase (DEHYDRO) (the peroxisomal bifunctional
protein) (Borges et al. 1993). Borges et al. (1993) suggested that PFDA’s inhibition ofthese peroxisomal enzymes and the resulting disruption of hepatic lipid cellular
metabolism may lead to the observed wasting syndrome.
Cytotoxicity
In HCT116 cells (a human epithelial-like colon carcinoma cell line), PFDA caused cell
death (Kleszczyński et al. 2007) and depolarization of plasma membrane potential in adose- and time-dependent manner (0, 100, 200, 300, or 400 µM, 4-72 hour incubation
period) (Kleszczyński and Składanowski 2009). In separate experiments, incubation of
HCT116 cells with PFDA resulted in kinase dephosphorylation (200 µM) (Kleszczyński
et al. 2009), acidification of the cyptoplasm (0-400 µM) (Kleszczyński and Składanowski2011), mitochondrial calcium overload (200 µM) (Kleszczyński and Składanowski 2011),
and reactive oxygen species (ROS) generation (at 200 µM) (Kleszczyński andSkładanowski 2009; Kleszczyński et al. 2009). At 200 µM PFDA, dissipation of
mitochondrial membrane potential led to ATP synthesis arrest, cytochrome c release,and activation of caspases associated with apoptosis (Kleszczyński et al. 2009).
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Effects on the hypothalamic-pituitary-gonadal-(liver) axis
The liver can be considered another component of the HPG axis in fish, as liverproduction of vitellogenin (Vtg), a precursor egg yolk protein, in male fish is under
estrogenic control (Jo et al. 2014). Studies investigating the effects of PFDA on
hormone levels, metabolism and receptor-mediated effects related to the HPGL axis are
summarized in Table 5.3.1 below.
Effects of PFDA on hormone levels have been studied in vivo in rats and zebrafish andex vivo in rats. In male rats treated with a single dose of PFDA there was a reduction inplasma levels of T and DHT at 40 and 80 mg/kg without effect on plasma
concentrations of LH (Bookstaff et al. 1990). However, in castrated male rats suppliedwith T via implant, a single dose of PFDA resulted in a decrease in seminal vesicle
weight at 20 and 40 mg/kg without an effect on ventral prostate weight or plasma Tlevels. In decapsulated testes from rats treated with a single dose of PFDA (40 or 80
mg/kg), there was a dose-related decrease in hCG-stimulated (100 mIU hCG/ml for 3
hours) T secretion (Bookstaff et al. 1990). Decreases in serum levels of T were also
observed in male SD rats exposed to PFDA for 28 days at 1.25 and 2.5 mg/kg-day(NTP 2019).
In male zebrafish, PFDA increased the plasma E2 to T ratio and the plasma E2 to 11ketotestosterone (11KT) ratio (Jo et al. 2014).
Effects of PFDA on hormone synthesis and metabolism have been studied in vivo inzebrafish and in vitro in a human placental choriocarcinoma cell line, in isolated rat
Leydig cells, and in a mouse Leydig tumor cell line. One study in zebrafish and one
study in a human cell line report that PFDA alters gene expression and enzyme activity,
respectively, of aromatase (CYP19A1), a steroidogenic enzyme that converts T to E2.
Specifically, a dose-dependent increase in aromatase gene expression (cyp19a) was
observed in male gonads of zebrafish exposed to PFDA (Jo et al. 2014), while adecrease in aromatase activity was observed in a human placental choriocarcinoma cellline (JEG-3 cells) following treatment with PFDA (Kjeldsen and Bonefeld-Jørgensen2013).
In isolated rat Leydig cells exposed in vitro at various concentrations for 48 h, treatment
with PFDA resulted in changes in hCG-stimulated T secretion, in the absence of
cytotoxicity or major morphological alterations (Boujrad et al. 2000). These changes
consisted of an increase in T production (e.g., secretion into the culture medium) at <10-5 M PFDA, and a decrease in T production at 10-5 M PFDA and above (Boujrad et al.
2000). In separate experiments in MA-10 cells (a mouse Leydig tumor cell line), PFDA
exposure decreased hCG-stimulated progesterone production (e.g., secretion into theculture medium) in a concentration- and time-dependent manner, in the absence of
cytotoxicity (Boujrad et al. 2000). In MA-10 cells PFDA also inhibited hCG-stimulated
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pregnenolone production and cAMP-stimulated progesterone production, decreasedmRNA and protein levels of TSPO, also referred to as peripheral-type benzodiazepine
receptor [PBR]) located on the outer mitochondrial membrane, and reduced the stabilityof TSPO mRNA (Boujrad et al. 2000). PFDA had no effect on P450scc (cyp11A1)
activity, steroidogenic acute regulatory protein (StAR) protein levels, mitochondrial
integrity, total protein synthesis, or DNA damage levels (assessed as poly(ADP-ribose)polymerase activity in MA-10 cells (Boujrad et al. 2000). The authors suggested that
decreased Leydig cell steroid synthesis following exposure to PFDA resulted from adecrease in transport of cholesterol from the outer to the inner mitochondrial membrane,
perhaps due to decreased outer membrane levels of TSPO (Boujrad et al. 2000).
In a mouse Leydig tumor cell line (mLTC-1 culture), incubation with PFDA inhibitedprogesterone production with an IC50 value of 11.52 µM (Zhao et al. 2017). The
authors suggested that PFDA disrupted progesterone production as a result of damage
to the mitochondrial membrane and increased production of ROS.
Hormone receptor mediated effects have been studied in vivo in fish and in vitro in ahuman embryonic kidney cell line, two human breast adenocarcinoma cell lines, and aChinese hamster ovary cell line after exposure to PFDA. In addition, hormone receptor
binding studies have been conducted with fish liver cytosol and modeled in silico. In
zebrafish, PFDA treatment increased liver gene expression of vitellogenin, an estrogen
responsive gene, in males (Jo et al. 2014). In rainbow trout, PFDA treatment via dietresulted in concentration-dependent increases in plasma levels of Vtg in studies with
11- and 5-month old juvenile trout (Benninghoff et al. 2011). In other experiments bythese authors, PFDA was shown to competitively bind (weakly) to trout liver ER (in liver
cytosol homogenates) (Benninghoff et al. 2011).
In human embryonic kidney (HEK293T) cells transfected with a hERα reporter gene,
PFDA (100-1000 nM) increased hERα reporter activity up to 2.5 fold (Benninghoff et al.
2011).
In MVLN cells (derived from MCF-7 cells, a human breast adenocarcinoma cell line,
stably transfected with a luciferase reporter gene and an estrogen-responsive elementfrom the Xenopus vitellogenin A2 gene), exposure to PFDA alone did not induce anestrogenic response. However, PFDA displayed anti-estrogenic activity in MVLN cells
pretreated with E2, inhibiting the estrogenic response to E2 (measured as luciferase
activity) in a concentration dependent manner. PFDA was a more potent inhibitor of the
estrogenic response than PFNA in these cells, with an EC50 of 20.3 µM (Juan Li et al.
2020). In separate experiments in MCF-7 cells, PFDA also had anti-estrogenic activity,with the addition of PFDA to E2-treated cells resulting in significant down regulation of
the E2-responsive genes TFF1 (trefoil factor 1, also known as pS2) and EGR3 (early
growth response 3) (Juan Li et al. 2020).
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Analyses using in silico computational models indicate that PFDA can efficiently dockwith human, mouse, and trout ERα in the ligand-binding domain and form a hydrogenbond at residue Arg394 in human ERα in a manner similar to that of estrogens(Benninghoff et al 2011). Additional in silico modeling studies with the activated form ofhERα, in which E2 is bound to the ligand-binding domain, predict that PFDA can bind tosites on the surface of the activated receptor (Juan Li et al. 2020).
PFDA significantly antagonized DHT-induced AR activity in a concentration-dependent
manner in a Chinese hamster ovary cell line (CHO-K1) transfected with AR and areporter vector (Kjeldsen and Bonefeld-Jørgensen 2013).

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Table 5.3.1 PFDA: Effects on the hypothalamic-pituitary-gonadal-(liver) axis in animals and in vitro studies5.
Reference; Experimental Model; N/group; Exposure Details

HPG-Related
Outcomes Assessed

Bookstaff et al. 1990

In vivo

In vivo

Plasma levels of T, DHT and LH

Adult male SD rats weighing approximately 295 g (10 per group).

Feed consumption and bodyweightTreatment: PFDA (96% purity) single i.p injection of 20, 40 or 80 mg/kg; two control groups:
vehicle and pair-fed vehicle controls. All animals were sacrificed on day 7 after dosing.
In a parallel experiment, rats (6 per group) were castrated and implanted with sustainedrelease testosterone-containing capsules two hours after receiving a single i.p injection of
PFDA at 20, 40 or 80 mg/kg, or vehicle (vehicle controls and pair-fed controls). Animals
were sacrificed on day 7 after dosing and the testes were used in the ex vivo experiments(summarized below).
Ex vivo

Ex vivohCG-stimulated T secretion,measured as the difference
between basal T secretion (inthe absence of hCG) and hCGstimulated secretion over a 3hour period

Incubation of decapsulated testis from adult rats 7 days after a single i.p. injection of 0, 20,
40, or 80 mg/kg PFDA (96% purity) in the absence or presence of 100 mIU hCG/ml for 3
hours.

Results
In vivo
Reduction in plasma levels of T and DHT at 40 and 80
mg/kg compared to vehicle and pair-fed controls. No effecton plasma concentrations of LH.
In castrated/testosterone-implanted rats PFDA treatment
had no effect on plasma T levels.
Reduced feed consumption and body weight at 80 mg/kg
compared to vehicle controls, and reduced body weight at
40 mg/kg compared to vehicle and pair-fed controls (on day
7 post-dosing).
Ex vivo
Dose-related decrease in hCG-stimulated T secretion by
decapsulated testes from rats treated with PFDA at 40 and
80 mg/kg, compared to testes from vehicle and pair-fed
controls.

NTP 2019

Serum T levels

Reduced serum T at 1.25 and 2.5 mg/kg-day

Male SD rats, 10-11 weeks old at the beginning of dosing, 10 rats per dose group.

Survival, body weightNo effect on survival. Lower body weights at ≥ 1.25 mg/kgday.

Treatment: PFDA (>97% purity, i.e., linear PFDA), once daily by gavage at doses of 0,0.156, 0.312, 0.625, 1.25, or 2.5 mg/kg-day, respectively, for 28 days.

Studies with in vivo experiments are presented first ordered by species (rat, mouse, fish), and within species, by publication date, followed bystudies with only in vitro experiements, ordered by publication date.
5

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Reference; Experimental Model; N/group; Exposure Details

HPG-Related
Outcomes Assessed

Results

Benninghoff et al. 2011

In Vivo

In Vivo

In vivo: Rainbow trout

Experiments 1 & 2: Vtg levels inplasmaExp.1: Concentration dependent increase in Vtg in plasma
levelsIn Vitro

Exp. 2: Concentration dependent increase in plasma Vtg
levels; LOEL: 0.64 ppm; NOEL: 0.128 ppm

Experiment 1: Dietary exposure of 11-month old juvenile rainbow trout to PFDA (highest
purity available) at
0, vehicle control (DMSO), 5, 50, or 250 ppm in Oregon Test Diet, OTD) 5 days/week for 14
days (equivalent to 0.1, 1, or 5 mg/kg-d, respectively); animals were sacrificed on day 15.
Positive control: 5 ppm E2

Trout liver cytosol: ERα binding
studies
HEK-293T cells: hERα genereporter activity

Trout liver cytosol: PFDA shows weak competitive binding toERα, completely displacing E2 from the receptor, with morethan a 10,000-fold lower relative binding affinity than E2.

In SilicoHEK-293T cells: PFDA at concentrations of 100-1000 nM
induced hERα gene reporter activity up to 2.5 foldN = 6 fish/treated and vehicle control (0.5 ppm DMSO) group.
N = 24 fish in untreated control group
Experiment 2: Dietary exposure of 5-month old juvenile rainbow trout to PFDA (highest
purity available) at
0, 0.026, 0.128, 0.64, 3.2,16, 80, 400, or 2000 ppm ppm in Oregon Test Diet, OTD) 5
days/week for 14 days; animals were sacrificed on day 15. Positive control: 5 ppm E2
N = 8 fish/treated group.
N = 16 fish/group in untreated controls and positive controls
In vitro: trout liver cytosol, HEK-293T cells

In Vitro

Molecular docking at the active
site in human, mouse and trout
ERα

In Silico
PFDA is predicted to bind at the active site of human, mouse,
and trout ERα. Predicted hydrogen bond interaction withhERα at Arg394.

Trout liver cytosol
ER binding studies in liver cytosol prepared from trout fed 5 ppm E2 (to induce ERα)HEK-293T cells: a human embryonic kidney cell line transfected with a human ERα
expression vector and a luciferase reporter plasmid containing an ERE.
PFDA or E2 treatment (1-1000 nM) for 24 hrs; hERα reporter gene activity measured.
In Silico
Molecular docking studies of PFDA with the active site in human, mouse, and trout ERα.

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Reference; Experimental Model; N/group; Exposure Details

HPG-Related
Outcomes Assessed

Results

Jo et al. 2014

In vivo

In vivo: zebrafish

PFDA (98% purity); Control dimethyl sulfoxide (DMSO).

Blood levels of E2, T, and 11KT.

No effect on blood levels of E2, T, or 11-KT, compared tocontrols. Increased ratio of E2/T and E2/11-KT at 1.0 mg/L,
compared to controls.

In vivo: zebrafish
Fertilized eggs exposed to 0, 0.01, 0.1, 1, or 10 mg/L PFDA until 120 dpf.

mRNA expression of genes inbrain, gonad, liver

N=3 /sex per tank were randomly sampled from four replicates.

In vitro

In vitro: H295R cells, a human adrenocortical carcinoma cell line

Levels of E2 and T

H295R cells were treated with PFDA at 0, 0.1, 1, 10, or 100 mg/L for 48 h.

Increased mRNA expression of cyp19b, erα, and er2β at 1
mg/L in male fish brain
Increased mRNA of cyp19a in male gonad and vtg1 in maleliver, both at 1 mg/L.
In vitro: H295R cells
No significant effects on E2, T, or E2/T ratio.

Boujrad et al. 2000

Isolated rat Leydig cells

Isoated rat Leydig cells

In vitro

Cell viability and morphology

PFDA inhibited hCG-stimulated T production, in the absenceof effects on cell viablilty or morphology.

Isolated rat Leydig cells:
Cells were isolated from adult SD (300 g) rats; 20–30% of the isolated cells stained positive
for the Leydig specific enzyme 3b-hydroxysteroid dehydrogenase. Cells were cultured for
48 h with PFDA (10-7 - 10-3 M) for 48 h, and then washed with serum free medium andstimulated for 2 hr with hCG (1 nM). hCG stimulated T production measured.
MA-10 mouse Leydig tumor cell line:
Cells wereexposed to PFDA (10-7 -3 x 10-3 M) for 24, 48, or 72 h. After 24, 48, or 72 hours,
cells were washed with serum free medium and stimulated for 2 h with hCG (1 nM).
Exp. 1: hCG stimulated progesterone production measured
Exp. 2: hCG stimulated pregnenlone production measured

T levels weremeasured inculture media after hCG
stimulation.
MA-10 mouse Leydig tumor
cellsExp. 1
Cell viability
Progesterone measured inculture media after hCG
stimulation
Exp. 2:Cell viability
Pregnenolone measured inculture media after hCG
stimulation
Protein determination (Westernblot)

MA-10 mouse Leydig tumor cells
Exp. 1:PFDA inhibited hCG-stimulated progesterone production in aconcentration-dependent manner, in the absence of effects
on cell viability.
Exp. 2:PFDA inhibited hCG-stimulated pregnenolone production, in
the absence of effects on cell viability. PFDA decreasedmRNA and protein levels of TSPO, and reduced the stabilityof TSPO.
No effects on CYP11A1 (P450scc) activity, StAR protein
levels, mitochondrial integrity, total protein synthesis, or DNA
damage (assessed as poly(ADP-ribose) polymerase activity

mRNA determination (Northern
blot)

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Reference; Experimental Model; N/group; Exposure Details
Kjeldsen and Bonefeld-Jørgensen 2013
In vitro: MVLN, CHO-K1 and JEG-3 cell lines
PFDA (97% purity) dissolved in dimethyl sulfoxide (DMSO), tested at concentrations of 10-9
– 10-4 M

HPG-Related
Outcomes Assessed
MVLN cells: ER transactivation
CHO-K1 cells: AR
transactivation
JEG-3 cells: Aromatase activity

Results
MVLN cells
Cytotoxic at 10-4 M
ER transactivation: No effects on ER activity
CHO-K1cells

MVLN cells: Derived from MCF-7 cells; stably transfected with a luciferase reporter gene
and an estrogen responsive element derived from the Xenopus vitellogein A2 gene.

Cytotoxic at 10-4 M

ER transactivation assessed with PFDA alone or co-treated with 25 pM E2

Concentration-dependent antagonistic effects on DHTinduced AR transactivation (IC50 6×10-5 M).

AR transactivation: No AR agonist activity.

CHO-K1 cells: Derived from CHO cells; transiently co-transfected with the AR plasmid anda luciferase reporter vector

JEG-3 cells

AR transactivation assessed with PFDA alone or co-treated with or 25 pM DHT

Cytotoxic at 10-4 M

JEG-3 cells: A human choriocarcinoma cell line. Aromatase activity assessed with PFDA
treatment

Decrease in aromatase activity down to 85 % at 10-5 M.

Zhao et al. 2017

Progesterone production

In vitro: Mouse Leydig cell tumor line (mLTC-1)

Mitochondrial membranepotential

PFDA (>95% purity)
Experiment 1: PFDA treatment (0-105µM) for 24 hrs; cAMP added and incubated for
additional 3 hrs; progesterone quantified via radioimmunoassay

Experiment 1: Concentration dependent decrease inprogesterone production (IC50 11.52 µM)
Experiment 2: Significant decrease in mitochondrial
membrane potential at 25 and 50 µM.

Experiment 2: PFDA treatment (0, 6.25, 12.5, 25, or 50 µM) for 24 hours; mitochondrial
membrane potential measured

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Reference; Experimental Model; N/group; Exposure Details
Juan Li et al. 2020
In vitro: MVLN and MCF-7 cell lines
PFDA (98% purity)
MVLN cells: Derived from MCF-7 cells; stably transfected with a luciferase reporter gene
and an estrogen responsive element derived from the Xenopus vitellogein A2 gene.
PFDA treatment (0, 0.0001-5 mM) for 48 hr in the presence or absence of E2 (1 nM) as
positive control or ICI 182,780 as negative control, or both; ER transactivation

HPG-Related
Outcomes Assessed
MVLN cells: ER transactivation
MCF-7 cells: mRNA gene
expression of estrogen
regulated genes TFF1/pS2,
EGR3
In silico: modeling predictions ofinteraction with E2-activatedhERαResults
MVLN cells
ER transactivation: No ER agonist activity with PFDA alone;in cells pretreated with E2 PFDA inhibited the estrogenic
response to E2 in a concentration dependent manner, with areported EC50 of 20.3 µM.
In cells cotreated with E2 and ICI 182,780 , PFDA caused asignificant increase in the inhibition of luciferase activity(attenuated luciferase response)

MCF-7 cells: human breast adenocarcinoma cell line

MCF-7 cells

PFDA treatment (50 µM) in the presence or absence of E2 for 48 hours; changes in
expression of estrogen regulated genes (TFF1/pS2, EGR3)
In silicomRNA levels of TFF1 and EGR3: No effect on gene
expression with PFDA alone. In cells cotreated with E2,PFDA significantly downregulated expression of both TFF1
and EGR3

Modeling analysis of PFDA interactions with E2-activated hERα

In silicoModeling predicts that PFDA can bind to the surface of theE2 activated form of hERα (at sites distinct from the ER
binding pocket, including the groove of co-activators)

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Effects on the thyroid
PFDA effects on thyroid hormones are summarized in Table 5.3. below.
Only one study (NTP 2019) provided data on outcomes of both thyroid toxicity and male
reproductive toxicity with repeated exposures to PFDA. Other available whole animalstudies (Gutshall et al. 1988; Gutshall et al. 1989; Langley and Pilcher 1985; Van
Rafelghem et al. 1987a) looked at aspects of thyroid toxicity following an acute dose of
PFDA. An in vitro study using rat pituitary GH3 cells evaluated PFDA for cytotoxicity,
effects on cell proliferation, and aryl hydrocarbon receptor (AhR) transactivity (Long etal. 2013). Cell-free biochemical assays and an in silico molecular docking simulation
were used to study binding affinities of PFDA to thyroid hormone transport proteins(Ren et al. 2016). Relevant information and data from these studies are summarized inTable 5.3..
The NTP (2019) 28-day study found decreased free thyroxine (T4) with PFDA doses >0.312 mg/kg-day. Total T4 (free plus bound), T3, and TSH were not affected. The same
animals showed effects on outcomes of male reproductive toxicity at doses > 1.25
mg/kg-day. Adverse outcomes included reduced testes and epididymal weights,
reduced serum testosterone, reduced epididymal sperm counts, and histopathology
indicative of interstitial testicular tissue atrophy, germ cell degeneration, and inhibited
spermiation.
Available whole-animal studies on the effects of PFDA on thyroid function used a single
dose at much higher levels than tested in the NTP (2019) 28-day study. None of thesestudies directly evaluated specific outcomes of male reproductive toxicity, but may help
interpretation by addressing a potential mechanistic pathway (Gutshall et al. 1988;
Gutshall et al. 1989; Langley and Pilcher 1985; Van Rafelghem et al. 1987a).
A single i.p. injection dose of 75 mg PFDA/kg to male rats was associated with reduced
serum total levels of T3, T4, and reverse triiodothyronine (rT3) at 12 and 24 hours postdosing (Gutshall et al. 1989). An in vitro binding experiment described in the same
paper reported that PFDA displaced radiolabeled T4 from binding sites on rat serum
albumin, which suggested a possible explanation for at least some of the decrease inserum T4 observed in vivo.
An earlier study by the same group (Gutshall et al. 1988) looked at the effects of 75 mg
PFDA/kg i.p., given with and without supplemental T4. On its own, 200 µg T4/kg
significantly increased serum total T4. When given in combination with PFDA,
supplemental T4 only partially restored total serum T4. The authors suggested theirresults could indicate an inability of PFDA-treated animals to retain T4 due to alterations
in plasma protein binding, increased metabolism, or increased excretion.
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Compared to ad libitum fed controls, significantly reduced body weight and feed intake
were seen in male rats given a single i.p. dose of PFDA at 40 or 80 mg/kg (p < 0.05 at
either dose) and evaluated seven days post-dosing (Van Rafelghem et al. 1987a). Eachdose group had their own pair-fed group and only those animals pair-fed to the 80
mg/kg dose group also had significantly reduced body weight compared to ad libitum
fed controls (p < 0.05). Total serum T4 was significantly reduced compared to ad libitum
controls with all three doses of PFDA (p < 0.05 at 20, 40, and 80 mg/kg). T4 levels forthe two lower dose PFDA groups were also significantly lower than their pair-fed
partners (p < 0.05); only the 80 mg/kg group and their pair-fed partners did not differ
from each other. Compared to ad libitum or pair fed controls, the 80 mg PFDA/kg group
had significantly higher serum T3 levels (p < 0.05), as well as lower T3 uptake (p < 0.05
for both comparisons). The authors concluded that despite observed effects of PFDA on
thyroid hormone levels, treated animals did not show a pattern of effects fully consistent
with hypo- or hyper-thyroidism.
The remaining in vivo study on thyroid effects of PFDA on male rats used a single i.p.
dose of 75 mg/kg, compared to vehicle and pair-fed controls (Langley and Pilcher1985). Serum total T4 levels were lower in PFDA-treated rats compared to pair-fed
controls at all tested times (p < 0.05 at 0.5, 1, 2, 4, 6, and 8 days post-treatment), andserum total T3 levels were significantly lower than for pair-fed controls at 0.5, 1, and 2days post-treatment (p < 0.05).
An in vitro study of PFDA effects on proliferation of T3-dependent rat pituitary GH3 cells
found no cytotoxicity under test conditions (Long et al. 2013). PFDA at 10−5 M
significantly decreased T3-induced cell proliferation (p < 0.05). In the absence of added
T3, PFDA alone at 1×10−8–1×10−5 M resulted in a concentration-dependent
decrease in GH3 cell proliferation (p < 0.05).
An additional experiment reported by Long et al. (2013) found no effect of PFDA
alone on aryl hydrocarbon receptor (AhR) transactivity. In the presence of TCDD,
1×10−9–1×10−7M PFDA further increased TCDD-induced AhR transactivity. The
authors noted consistency of their results with a potential for cross-talk between AhR
and peroxisome proliferator-activated receptor alpha (PPARα). PFDA is a known
PPARα agonist.
With binding of T4 to the thyroid transport protein transthyretin (TTR) representing a
relative potency (RP) of 1, the RP for PFDA binding to TTR was 0.019, as compared to
0.083 for PFOA (Ren et al. 2016). For wild type thyroxine-binding globulin (TBG), an RP
was not detectable for PFDA. In the molecular docking experiment with TTR, the longer
fluorinated carbon chain structure of PFDA was nearly too large, and required thefluorinated carbon tail to bend so that the ligand could fit into the binding pocket.

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Weiss et al. (2009) tested PFDA, as well as other related compounds, for binding tohuman TTR in competition with 125I-labeled T4. PFDA inhibited 46% of T4-TTR binding.
Overall, studies of the mechanisms by which PFDA interferes with normal thyroid
hormone function suggest interference with binding of T4 to transport proteins (Gutshallet al. 1988; Gutshall et al. 1989; Ren et al. 2016). Such interference could result inalterations to normal thyroid hormone transport, metabolism, and feedback systems, butdoes not necessarily explain all manifestations of PFDA toxicity (Van Rafelghem et al.
1987a). The only study which directly evaluated effects of PFDA on outcomes of male
reproductive toxicity as well as on outcomes of thyroid toxicity was the NTP (2019)study. At the two highest doses, reduced levels of free T4 and increased adverse
effects on male reproductive outcomes co-occurred. There are some similarities
between the types of male reproductive effects reported with PFDA treatment (NTP2019) and those reported to occur with post-pubertal hypothyroidism (Alahmar et al.
2019; Krajewska-Kulak and Sengupta 2013). However, while the evidence is consistent
with a possible mechanistic connection between alterations in thyroid hormones and themale reproductive effects of PFDA, based on the mechanistic evidence alone, a causal
connection is far from established.

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Table 5.3.2 .PFDA: Effects on thyroid hormones 6.
Study Design
Langley and Pilcher 1985
Male Wistar rats (200-250 g) given single i.p.
injection of PFDA at 75 mg/kg (N = 30)
One day after above, 30 weight-matched malerats were given propylene glycol vehicle and pair
fed to treated rats.
Additional vehicle controls (N = 8) were pair fed
to treated group.
At intervals starting 12 hrs post treatment, treatedand control rats sacrificed for blood collection.

Outcomes Assessed

Major Findings

Daily food consumption, body weight,
and rectal temperature for 8 days.

Body weights of PFDA-treated rats fell from pre-treatment
average of 250 g to 160 by 8 days post-treatment.

Resting heart rates measured every
other day starting 2 days following
treatment.

Body weights of pair-fed rats fell to a final weight of 190 g.
Ad lib-fed rats gained to a final weight of roughly 275 g.

Measurements of total T4 and total T3
from samples collected at staggered
sacrifice (0.5, 1, 2, 4, 6, and 8 days
following treatment).

Comments

Compared to pair-fed controls, body temperature in PFDAtreated rats was significantly increased (p < 0.05) on the day
following treatment, but significantly decreased (p < 0.05) at
3, 4, 5, 6, 7, and 8 days following treatment.
Resting heart rate progressively slowed in PFDA-treatedrats, reaching statistical significance on days 6 and 8
following treatment (p < 0.05). Resting heart rate of pair-fed
controls was not affected.
Serum T4 levels were lower in PFDA-treated than pair-fed
control at all tested times (p < 0.05). Results for T3 were“similar but less dramatic,” significantly (p < 0.05) lower thanfor pair-fed controls at 0.5, 1, and 2 days post treatment.

Van Rafelghem et al. 1987a

Daily body weights.

Young adult male Sprague-Dawley rats given asingle dose, i.p. of PFDA at 0, 20, 40, or 80
mg/kg.

Body temperature measured at 7 days
post PFDA or vehicle treatment.

Each treated rat had a pair-fed “partner”; pair-fed
and ad-lib fed controls given vehicle (1 ml/kg
propylene glycol and water, i.p.).
Evaluation at 7 days post-dosing.

6

Absolute and relative total oxygen
consumption at 7 days post treatment;
measured during periods of bothmotor activity and rest for 10 out of
every 30 minutes over 4 hours. Data
were presented as both absolutedeterminations and as relative to“metabolic body size”.

Reduced feed intake and body weight (p < 0.05 for 40 and
80 mg PFDA/kg for both outcomes).
Body weight was reduced in animals pair fed to the 80 mg/kg
dose group (p < 0.05).
Thyroid gland weight for the 80 mg PFDA/kg group was
significantly lower than either pair fed or ad lib fed controls (p< 0.05 for both comparisons). Thyroid weight for the grouppair fed to the 80 mg/kg group was significantly lower thanthyroid weights for ad lib fed controls (p < 0.05).

Studies are ordered by species (in vivo followed by in vitro) and within species, by publication date.

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Study Design

Outcomes Assessed

Major Findings

Resting oxygen consumption (total
and relative) was also determined foranimals inactive for at least 5 minutes
prior to measurements.

Serum total T4 showed a dose-related decrease. Levels
were significantly below ad lib controls for all doses (p <0.05). T4 levels with 20 or 40 mg PFDA/kg were significantly
lower than for their corresponding pair-fed group (p < 0.05).
The 80 mg/kg group was not significantly different from pairfed controls.

Basal metabolic rate was determinedfrom oxygen consumption corrected
for metabolic body size.
Total T4 and T3 concentration inplasma.
Unsaturated binding capacity ofthyroid-binding proteins was assessedby T3 uptake.
Free thyroxine index.
Thyroid gland weights and histology.

Comments

Serum T3 levels for the 80 mg/kg group were significantly
higher than either pair fed or ad lib fed controls (p< 0.05).
Pair fed controls for the 80 mg/kg group had significantly
lower T3 than the ad-lib controls (p < 0.05).
T3 uptake was significantly lower in the 80 mg/kg group thanfor either pair fed or ad lib controls (p < 0.05). T3 uptake was
not different for pair fed and ad lib controls.
Body temperature of 80 mg/kg group was significantly lower
than controls (p < 0.05), while pair fed animals were notaffected.
Absolute total oxygen consumption was significantly reduced
for both the 80 mg PFDA/kg group and their pair fed controls(p < 0.05). Relative oxygen consumption was not
significantly affected for any group.
Resting oxygen consumption was significantly reduced fromcontrols for the 80 mg/kg group and their pair fed controls –for both absolute and relative determinations (p < 0.05).
Basal metabolic rate reduced with PFDA (8% at 80 mg/kg),
but even more for pair fed animals (18%).
PFDA caused a dose-dependent decrease in thyroid glandweight (not paralleled by pair feeding alone). No effect on
thyroid histology.

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Study Design
Gutshall et al. 1988
Male Wistar rats (175-225 g; 8-12/group) given:0, 50, 100, 200, or 250 µg/kg of T4, i.p. daily for
7 days prior to a single acute dose of PFDA i.p.
at 75 mg/kg

Outcomes Assessed
Body wt, food consumption, rectal
body temperature. Total feces dry
wt/day.
Blood collected at sacrifice for thyroid
hormone collection.

Major Findings
200 µg T4/kg reduced “hypophagia” induced by 75 mg
PFDA/kg.
Other doses of T4 less effective or ineffective.
T4 treatment did not prevent PFDA-induced body weight
loss, even where food consumption was similar to controls.

0 or 200 µg/kg of T4, i.p. daily for 7 days prior toa single acute dose of PFDA i.p. at 75 mg/kg

PFDA-induced hypothermia was not alleviated bysupplemental T4.

T4 treatment was continued daily until sacrifice
14 days after PFDA dose.

PFDA alone: total T4 lower than in control or PFDA + T4
groups (p < 0.05 for both). Serum T3 levels were notaffected.

Comments
PFDA “wasting syndrome” could not
be entirely attributed to hypophagia,
or to reduced serum thyroid
hormones.
PFDA-induced reductions in T4,
even when supplemented, may
indicate inability to retain T4 due toaltered plasma protein binding, orincreased metabolism or excretion.

200 µg T4/kg alone caused significant elevations in serum
T4 and T3.
T4 levels in the PFDA + T4 group were higher than for PFDA
alone, but significantly lower than for untreated controls
Gutshall et al. 1989
Male Wistar rats (150-250 g); N=5; single i.p.
injection dose of 75 mg PFDA/kg, pair fed vehicle
controls and untreated ad libitum fed controls.
Radioiodine uptake experiment:
Rats dosed with PFDA or vehicle,
Nine hours later given 14 mg propylthiouracil(PTU) sub cutaneous injection.
One-half to one hour after PTU, 5 µCi Na125I.
Blood drawn for analysis two hours following
radioiodine.
500 µg synthetic thyrotropin-releasing hormone(TRH)/kg-bw by i.p. injection 22 hours following
PFDA or vehicle. Sacrifice 24 hours following
PFDA.
In vitro effect of varying concentrations of PFDA
on binding of [125I]T4 to rat albumin at pH 7.4, 30
min, at 37°C.

PFNA and its salts andPFDA and its salts

Food consumption and body weightrecorded daily.

Trapping of radiolabeled iodine by thyroid gland unaffectedby PFDA.

Radioiodine uptake.

PFDA:

Response to TRH challenge.

- depressed response to a TRH challenge, suggesting
possible function lesions in the thyroid and/or pituitary
glands (p < 0.05 for both T3 and T4 at baseline and 2 and 4
hours post challenge).

Serum levels of reverse
triiodothyronine (rT3)
Changes in activity of thyroid
hormone-sensitive liver enzymes in
response to PFDA
Liver mitochondrial α-glycerol
phosphate dehydrogenase (GPD) andcytosolic malic enzyme (ME)activities.
In vitro albumin binding of thyroxine(T4) in the presence of varying
concentrations of PFDA.

Reduced serum thyroid hormone
levels seen in PFDA-treated rats may
result from reduced responsivenessof the pituitary and/or thyroid glands
to hormonal stimulation, and/or
displacement of hormones fromplasma protein binding sites.

- decreased serum T3, T4, and rT3 (p < 0.05 at 12 and 24
hours post treatment), indicating reduced serum T4 not due
to enhanced conversion to T3.displaced radiolabeled T4from binding sites on serum albumin, which may partiallyaccount for decrease in serum T4.
- caused significant increases in GPD and ME activity(p < 0.05 for both enzymes at 7 and 14 days, but not 24
hours).
Effects further increased by T4 pretreatment (p < 0.05 forboth enzymes at 24 hours, 7 and 14 days post PFDA). T4 +PFDA treatment enhanced the effects of PFDA on these
enzymes with both stronger and earlier increases (p < 0.05
at all time-points).

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Study Design

Outcomes Assessed

Major Findings

Comments
In the same study, direct evidence ofmale reproductive toxicity wasreported at doses > 1.25 mg/kg-day.
Outcomes included reduced testis
and epididymis weights, reduced
serum testosterone levels, reduced
epididymal sperm counts, increased
incidence of interstitial tissue
atrophy, germ cell degeneration, andinhibited spermiation.

NTP 2019 (see Table 5.2.1 for summary of male
reproductive toxicity data from this study)

Body weights and thyroid glandweights and histology.

No effect on absolute thyroid gland weight; increased relative
thyroid gland weight.

Male SD rats, 10-11 weeks old, 10 rats/dose.
PFDA administered once daily by gavage indeionized water with 20% Tween® 80 at doses of0, 0.156, 0.312, 0.625, 1.25, or 2.5 mg /kg-day,for 28 days.

Blood levels of TSH, T3, and T4 (total
and free).

No effect on TSH, T3,or total T4% inhibition of T4-TTR binding at
maximum concentration,
concentration at 50% inhibition (IC50),
slope of dose-response curve, relative
potency compared to T4 (T4-RP
factor).

For PFDA:

Decreased free T4 at PFDA doses > 0.312 mg/kg-day (p <0.01).

At the end of the study, blood was collected fromthe abdominal aorta for biochemical analysis(including thyroid stimulating hormone (TSH) andthyroid hormones.
Weiss et al. 2009
Cell-free biochemical assay:
PFDA (≥98% purity) tested for binding to human
TTR in competition with 125I-labeled T4.

Long et al. 2013
Two in vitro tests with PFDA (98% purity):
3.
4.

Proliferation of T3- dependent rat pituitary
GH3 cells using the T-screen assay
Effect on aryl hydrocarbon receptor (AhR)
transactivation in the AhR-luciferase
reporter gene bioassay in transfectedmouse Hepa1.1 2cR cells.

46% inhibition of T4-TTR binding at maximum concentration
IC50 = 8954 nM (compared to 61 nM for T4)
Slope = -1.65 (compared to -1.17 for T4)
T4-RP factor = 0.007 (T4 = 1)

Cytotoxicity to rat pituitary GH3 cells
and transfected mouse cells

PFDA was not cytotoxic to rat pituitary GH3 cells ortransfected mouse cells under test conditions.

Proliferative response of GH3 to
PFDA or T3 alone, or PFDA in thepresence of 0.5 nM T3.

Concentration-dependent increase in GH3 cell proliferation
with T3 alone at 1×10−10–1×10−8 M.

Effect on AhR transactivity of PFDA in
the absence or presence of 60 pM
2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD).

Concentration-dependent decrease in GH3 cellproliferation with PFDA alone at 1×10−8–1×10−5 M (p <0.05).
PFDA at 10−5 M significantly decreased T3-induced cellproliferation (p < 0.05).

Binding potency of PFASs decreasedin the order: PFHxS > PFOS/PFOA>
PFHpA > sodium perfluoro-1octanesulfinate > PFNA, with TTR
binding potencies 12.5–50 times
lower than T4.
AhR upregulates phase I
metabolizing enzymes such as arylhydrocarbon hydroxylase.
Study authors note consistency of
their results with previous
suggestions of a potential for crosstalk between AhR and PPARα.
PFDA is a known PPARα agonist inmouse cells.

PFDA alone had no effect on AhR transactivity intransfected mouse cells.
1×10−9–1×10−7 M PFDA in the presence of TCDD further
increased TCDD-induced AhR transactivity to 124% of 60
pM TCDD.

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Study Design
Ren et al. 2016
Cell-free biochemical assays:
Quantitative assessment of binding affinities of
PFASs (including PFDA, purity not specified) tohuman TTR and human thyroxine-binding protein(TBG) by a fluorescent competitive binding
assay.
Molecular docking (in silico) was used to simulateinteraction of PFDA with TTR.

PFNA and its salts andPFDA and its salts

Outcomes Assessed

Major Findings

Comments

Binding affinities with thyroid hormone
transport proteins: TTR and TBG.

The relative potency (RP) for PFDA binding with TTR was
0.019, as compared to T4 representing an RP of 1 and 0.083
for PFOA.

The molecular docking model
showed that T4 could fill the TTR
ligand-binding pocket. PFOA andPFOS could also fill the pocket, withthe fluorinated carbon tail extended.
Perfluroalkyl acids with fewer than 8chained carbons were too small to fill
the pocket, while longer fluorinatedcarbon chains required folding of thetail in order to fit the pocket.

Molecular docking of PFDA with TTR.

For TBG, binding was not detectable for PFDA or PFOA.
In the molecular docking experiment with TTR, the longer
fluorinated carbon chain structure of PFDA was nearly toolarge, and required the fluorinated carbon tail to bend so thatthe ligand could fit into the binding pocket.

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Possible involvement of peroxisome proliferator-activated receptors
PPARs are lipid-activated transcription factors that belong to the nuclear hormone
receptor family. There are three isotypes of PPARs, each of which are expressed inrodents and humans: PPARα, PPARβ (also known as δ, NUC-1 or FAAR), and PPARγ.
They are differentially expressed during rat embryonic development and expression
levels can fluctuate during different periods of development (Braissant and Wahli 1998).
The three PPAR isoforms are expressed in rat testis, mostly in Leydig and Sertoli cells,
and in human Leydig cell tumor cells (Kotula-Balak et al. 2020; Michalik et al. 2002).
Natural ligands of PPARs include some fatty acids and eicosanoids. A number ofxenobiotic chemicals are PPAR agonists, including PFDA and other PFASs (e.g.
PFNA).
Although PFDA has been demonstrated to be an agonist of PPARα (NTP 2019), 2019),
there is little evidence to suggest that PPAR activation is involved in the effects of PFDA
on the testes. The findings from a series of 28-day studies in male rats conducted by
NTP of four perfluoroalkyl carboxylates (perfluorohexanoic acid [PFHxA], PFOA, PFNA,
PFDA) and one PPARα agonist (Wyeth-14643) indicate that PPARα activation isunlikely to be involved in the testicular toxicity observed following treatment with PFDA
(See Section 5.2 and Tables 5.2.1 and 5.2.2) (NTP 2019). Specifically, liver effects
consistent with PPARα activation were observed with Wyeth-14643 and all four
perfluoroalkyl carboxylates, however, only treatment with PFDA or PFNA resulted in
obvious testicular damage, suggesting PFDA and PFNA target the testis through amechanism that does not involve PPARα activation (NTP 2019).
5.4 PFDA: Summary of evidence on male reproductive toxicity
Human studies
The epidemiologic evidence for an effect of PFDA on AGD was mixed, with the strongerof two studies reporting an association with shorter AGDAS and AGDAP that did not
remain at 12 months of age (Tian et al. 2019).
Higher serum PFDA was associated with lower serum testosterone in a small sample ofadolescent boys (Zhou et al. 2016), while studies of adult males did not reportconsistent associations with reproductive hormones (Joensen et al. 2009; Joensen et al.
2013; Ma et al. 2021; Petersen et al. 2018).
Semen quality studies with the highest PFDA concentrations reported someassociations with poorer semen quality. The sample in the study by Pan et al. (2019)was the largest (N=664), overrepresented infertile men, and measured both serum and
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semen PFDA. This study reported that semen PFDA was associated with decreased
progressive motility and VSL and with higher HDS and DFI, and that serum PFDA wasalso associated with HDS (Pan et al. 2019). In a small cohort of couples undergoing
IVF due to female factor infertility, plasma PFDA in male partners was associated withdecreased sperm concentration and possibly reduced count (Ma et al. 2021).
One study examined PFDA and prostate cancer and reported an association among
men with a first-degree relative with prostate cancer; this result needs corroboration(Hardell et al. 2014).
Animal studies
PFDA reduced testis weight in rats (in two studies), hamsters and guinea pigs following
a single i.p. injection at relatively high doses ranging from 50-175 mg/kg (George andAndersen 1986; Olson and Andersen 1983; Van Rafelghem et al. 1987b). In an oral
study, dose-related reductions in testis and epididymis weights were observed in rats
exposed to lower doses for 28 days (≥1.25 mg/kg-d) (NTP 2019).
Histopathological changes in the testis (i.e., germ cell degeneration) were observed inrats (in two studies), hamsters, and guinea pigs that received a single i.p. dose of ≥50
mg/kg (or 150 mg/kg for guinea pigs) (George and Andersen 1986; Olson and Andersen
1983; Van Rafelghem et al. 1987b). Testicular lesions observed in rats exposed tolower doses for 28 days included a dose-dependent increase in interstitial cell atrophy(≥1.25 mg/kg-day), and increased spermatid retention or inhibited spermiation (2.5mg/kg-day) (NTP 2019). No effects on testis weight or histology were observed in miceon day 28 following a single i.p. dose of 250 mg/kg PFDA.
The one study that measured sperm parameters found a dose-dependent reduction in
epididymal sperm counts in rats exposed to PFDA for 28 days, which was significant at
2.5 mg/kg-day (NTP 2019). Reduced serum levels of T were observed in rats exposed
to PFDA for 28 days (dose-dependent; statistically significant at ≥1.25 mg/kg-day) (NTP2019) and in rats receiving a single dose (≥40 mg/kg) (Bookstaff et al. 1990). Plasma
DHT levels were also decreased by PFDA in the single dose rat study by Bookstaff et
al. 1990. The ratios of blood E2/T and E2/110-KT were increased in zebrafish exposedfrom 1-120 dpf to PFDA at 1.0 mg/L, but not at higher or lower concentrations (Jo et al.
2014). There are no animal studies available on the male fertility effects of PFDA.
Coherence of results in human and animal studies
Findings from a limited number of epidemiological studies identified associations ofincreased serum levels of PFDA with reduced sperm count and poor sperm motility.
These effects are consistent with reduced epididymal sperm counts observed in rats
following 28-day oral treatment with 2.5 mg/kg-day PFDA (NTP 2019), and with germ
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cell degeneration observed in rats, hamsters, and guinea pigs treated with a single,
relatively high dose of PFDA.
Higher serum PFDA was significantly associated with lower serum T in a small sample
of adolescent boys (Zhou et al. 2016), but not in four studies of adult men. Reduced
serum T was also observed in rats following 28-day oral treatment with PFDA at dosesof ≥1.25 mg/kg-day (NTP 2019) and in rats treated with a single i.p. injection at 40 or 80
mg/kg (Bookstaff et al. 1990).
Mechanistic considerations
While a number of mechanistic pathways could potentially contribute to PFDA-induced
male reproductive effects, direct mechanistic relationships to apical outcomes have notbeen established. Potential mechanistic pathways involved in PFDA mediated male
reproductive toxicity include effects on the HPG axis, and effects on thyroid
homeostasis.
HPG axis
Whole animal, ex vivo, and in vitro studies indicate PFDA affects steroid hormone levels(Bookstaff et al. 1990; Jo et al. 2014) and hormone synthesis and metabolism (Boujrad
et al. 2000; Jo et al. 2014; Kjeldsen and Bonefeld-Jørgensen 2013; Zhao et al. 2017).
Reduced serum levels of T were observed in rats exposed to PFDA for 28 days atdoses of 1.25 and 2.5 mg/kg-day (NTP 2019). In male rats treated with a single dose of
40 or 80 mg/kg-day, PFDA reduced plasma levels of T and DHT without effects on
plasma levels of LH and caused a dose-dependent decrease in hCG-stimulated T
secretion in decapsulated testes from PFDA treated animals, indicating disruption of
testicular feedback to LH stimulation (Bookstaff et al. 1990). In isolated rat Leydig cells
and mouse Leydig tumor cell lines, exposure to PFDA resulted in decreases in steroid
hormone production (Boujrad et al. 2000; Zhao et al. 2017). Studies in mouse Leydig
tumor cell lines found that PFDA reduced TSPO protein levels and stability of TSPOmRNA and decreased mitochondrial membrane potential, suggesting that impairment ofcholesterol transport within the mitochondria, and altered mitochondrial function related
to decreased membrane potential may also contribute to decreases in steroidogenesis(Boujrad et al. 2000; Zhao et al. 2017). PFDA may also affect the rate of conversion of T
to E2, as it increased gonadal aromatase gene expression in male zebrafish (Jo et al.
2014), while it decreased the level of aromatase activity in a human placental
choriocarcinoma cell line (Kjeldsen and Bonefeld-Jørgensen 2013).
There is evidence that PFDA can interact with estrogen receptors from receptor binding
studies (Benninghoff et al. 2011), and this is supported by predictions from in silico
modeling studies (Benninghoff et al. 2011; Juan Li et al. 2020). Estrogen receptor
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mediated effects of PFDA have been observed in fish (Benninghoff et al. 2011; Jo et al.
2014) and human cell lines (Benninghoff et al. 2011; Juan Li et al. 2020). In fish, vtg
gene expression was increased in zebrafish (Jo et al. 2014) and Vtg protein levels wereincreased in trout (Benninghoff et al. 2011). PFDA induced an estrogenic response in
human embryonic kidney cells transfected with an hERα reporter gene (Benninghoff et
al. 2011), while anti-estrogenic activity was observed in studies with human breast
adenocarcinoma cell lines (Juan Li et al. 2020).
PFDA may also interact with androgen receptors, based on observations of anti-AR
activity in a Chinese hamster ovary cell line (Kjeldsen and Bonefeld-Jørgensen 2013).
Thyroid homeostasis
PFDA, like many PFASs, can disrupt thyroid homeostasis and this may play a role inmale reproductive toxicity (NTP 2019; Rajender et al. 2011; Ren et al. 2016; Weiss etal. 2009; Xie et al. 2020). Whole-animal and in vitro studies indicate PFDA interfereswith normal thyroid hormone function (Gutshall et al. 1988; Gutshall et al. 1989; Langley
and Pilcher 1985; Long et al. 2013; NTP 2019; Ren et al. 2016; Van Rafelghem et al.
1987a), more specifically with the binding of T4 to transport proteins (Gutshall et al.
1988; Gutshall et al. 1989; Ren et al. 2016). At the two highest PFDA doses tested in
the NTP (2019) study, reduced levels of free T4 and increased adverse effects on male
reproductive outcomes co-occurred. While there are some similarities between thetypes of male reproductive effects reported with PFDA treatment in rats (NTP 2019) andthose reported to occur in humans with post-pubertal hypothyroidism (Alahmar et al.
2019; Krajewska-Kulak and Sengupta 2013), a clear mechanistic connection between
the effects of PFDA on thyroid hormones leading to male reproductive effects has notbeen established.
Key characteristics of male reproductive toxicants and endocrinedisrupting chemicals
Recently a set of key characteristics (KCs) that are frequently exhibited by exogenous
agents that cause male reproductive toxicity was identified, based on a survey of known
male reproductive toxicants and established mechanisms and pathways of toxicity(Arzuaga et al. 2019). The KCs can encompass many types of mechanistic endpoints
and are not constrained to previously formulated hypotheses, allowing a broader
consideration of multiple mechanistic pathways and hypotheses. The eight KCs of male
reproductive toxicants, have been presented earlier in this document, in Section 4.4(See Table 4.4.1). A chemical need not exhibit all of the KCs, and is not expected to
exhibt all of the KCs, in order to be considered a male reproductive toxicant.

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Another set of KCs has been developed for EDCs, based on knowledge of hormone
actions and endocrine-disrupting chemical effects (La Merrill et al. 2020). Because
endocrine-disrupting chemicals may be male reproductive toxicants, the set of ten KCsof endocrine-disrupting chemcals has also been presented earlier in this document, inSection 4.4 (See Table 4.4.2). As is true for the KCs of male reproductive toxicity, achemical need not exhibit all of the KCs, and is not expected to exhibt all of the KCs, inorder to be considered an endocrine-disrupting chemical.
The available studies on PFDA and its salts provide evidence related to four KCs ofmale reproductive toxicants, and four KCs of endocrine disrupting chemicals. This
information is briefly summarized below. There is clear overlap among KC3 of male
reproductive toxicants (Male KC3) and KC8 of endocrine-disrupting chemicals (EDC KC
8), where both refer to an effect on hormone levels, with Male KC3 being specific toeffects on reproductive hormone levels. The data relevant to effects on reproductive
hormone levels is therefore presented as being related to both of these KCs.
Male KC1. Alters germ cell development, function, or death
Two studies in humans reported associations of PFDA with effects on sperm function or
count. PFDA was associated with decreased progressive motility and VSL in one study(Pan et al. 2019), and with decreased sperm concentration in another (Ma et al. 2021).
In the one animal study that assessed parameters related to sperm quality, reduced
epididymal sperm count was observed in SD rats exposed to PFDA for 28 days (NTP2019). Histopathological changes in the testis (i.e., germ cell degeneration) wereobserved in three studies in rats (NTP; 2019; George and Andersen 1986; Olson andAndersen 1983) and one study each in hamsters, and guinea pigs (Van Rafelghem etal. 1987b).
Male KC2. Alters somatic cell development, functions, or death
A dose-dependent increase in testicular interstitial cell atrophy was reported in a 28-day
study in rats (NTP 2019). Other studies in animals have reported atrophy anddegeneration of the seminiferous tubules in Fischer rats (George and Andersen 1986),
in golden hamster, and guinea pigs (Van Rafelghem et al. 1987b).
Male KC3: Alters production and levels of reproductive hormones;
EDC KC8: Alters hormone distribution or circulating hormone levels
In whole animals and in vitro, there are several studies showing effects on hormonallevels. PFDA reduced serum or plasma levels of T in rats (NTP 2019, Bookstaff et al.
1990), and reduced production of T in decapsulated rat testis from PFDA- treatedanimals (Bookstaff et al. 1990), and in isolated rat Leydig cells (Boujrad et al. 2000).
Decreased hCG-stimulated progesterone and pregnenolone production in MA-10 cells
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(Boujrad et al. 2000) and in mLTC-1 culture (Zhao et al. 2017) as well as increases in
the plasma E2 to T ratio and plasma E2 to 11KT ratio in male zebrafish (Jo et al. 2014)
have also been reported.
Male KC4. Alters hormone receptor levels/functions and
EDC KC2. Antagonizes hormone receptors
Studies are available on PFDA and its potential to affect hormone receptors. PFDA has
been shown to increase hERα reporter activity in HEK293T cells (Benninghoff et al.
2011); show antagonistic effects on DHT-induced AR transactivation in CHO-K1 cells(Kjeldsen and Bonefeld-Jørgensen 2013); cause suppression of luciferase induction by
E2 (in a concentration-response manner), and result in significant down regulation inlevels of estrogen responseive genes in MVLN cells (Juan Li et al. 2020).
EDC KC1. Interacts with or activates hormone receptors
Interaction with hormone receptors include increased liver gene expression of vtg, anestrogen responsive gene, in male zebrafish (Jo et al. 2014) and plasma levels of Vtg inmale rainbow trout (Benninghoff et al. 2011). PFDA showed weak competitive binding
to ER in trout liver cytosol homogenates (Benninghoff et al. 2011); and antagonistic
effects on DHT-induced AR transactivity in CHO-K1 cells (Kjeldsen and BonefeldJørgensen 2013). In a MVLN cell line, PFDA suppressed luciferase induction by E2 in aconcentration-response manner (Juan Li et al. 2020), suggesting interaction with theER.
EDC KC4 Alters signal transduction in hormone- responsive cells factors andtranscripts, and activity
In a MVLN cell line, PFDA caused a significant downregulation in estrogen responsive
genes (tff1 and egr3) in the presence of E2 (Juan Li et al. 2020).
EDC KC 6. Alters hormone synthesis
Some in vitro studies have reported effects on hormone synthesis. These include
decreased mRNA and protein levels of TSPO located on the outer mitochondrial
membrane, reduced the stability of TSPO mRNA In MA-10 cells and a reduction in T
production in isolated rat Leydig cells (Boujrad et al. 2000). Additionally, a dosedependent increase in aromatase gene expression (cyp19a) in male gonads ofzebrafish (Jo et al. 2014) and a decrease in aromatase activity in JEG-3 cells (Kjeldsen
and Bonefeld-Jørgensen 2013) were reported.
EDC KC8. Alters hormone distribution or circulating hormone levels
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In addition to effects on reproductive hormones (discussed above in conjuction withMale KC8), alterations in other hormones reflective of EDCs were noted for PFDA inanimal models both in vivo and in vitro. Reduced serum T4 levels in male Wistar rats
(Gutshall et al. 1988; Langley and Pilcher 1985) and in male SD rats (NTP 2019);
reduced serum T3 and T4 in male SD rats (Van Rafelghem et al. 1987a) and T3, T4,
and rT3 in male Wistar rats (Gutshall et al. 1989); as well as inhibition of T4-TTR
binding in a cell-free biochemical assay (Weiss et al. 2009) have been noted.
EDC KC10. Alters fate of hormone- producing or hormone-responsive cells
Decreased thyroid gland weight in male SD rats (Van Rafelghem et al. 1987a) and aconcentration-dependent decrease in GH3 cell proliferation in vitro (Long et al. 2013)
were also observed.

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neonatal mice testes. J Appl Toxicol 39:1663-1671.
Specht IO, Hougaard KS, Spanò M, Bizzaro D, Manicardi GC, Lindh CH, et al. 2012.
Sperm DNA integrity in relation to exposure to environmental perfluoroalkyl substances

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- a study of spouses of pregnant women in three geographical regions. Reprod Toxicol
33:577-583.
Tian Y, Liang H, Miao M, Yang F, Ji H, Cao W, et al. 2019. Maternal plasma
concentrations of perfluoroalkyl and polyfluoroalkyl substances during pregnancy andanogenital distance in male infants. Hum Reprod 34:1356-1368.
Toft G, Jönsson BA, Lindh CH, Giwercman A, Spano M, Heederik D, et al. 2012.
Exposure to perfluorinated compounds and human semen quality in arctic andeuropean populations. Hum Reprod 27:2532-2540.
Van Rafelghem MJ, Inhorn SL, Peterson RE. 1987a. Effects of perfluorodecanoic acidon thyroid status in rats. Toxicol Appl Pharmacol 87:430-439.
Van Rafelghem MJ, Mattie DR, Bruner RH, Andersen ME. 1987b. Pathological andhepatic ultrastructural effects of a single dose of perfluoro-n-decanoic acid in the rat,
hamster, mouse, and guinea pig. Fundam Appl Toxicol 9:522-540.
Vanden Heuvel JP, Kuslikis BI, Van Rafelghem MJ, Peterson RE. 1991. Disposition of
perfluorodecanoic acid in male and female rats. Toxicol Appl Pharmacol 107:450-459.
Vanden Heuvel JP, Kuslikis BI, Peterson RE. 1992. Covalent binding of perfluorinatedfatty acids to proteins in the plasma, liver and testes of rats. Chem Biol Interact 82:317328.
Wang Y, Zhong Y, Li J, Zhang J, Lyu B, Zhao Y, et al. 2018. Occurrence of
perfluoroalkyl substances in matched human serum, urine, hair and nail. J Environ Sci(China) 67:191-197.
Weiss JM, Andersson PL, Lamoree MH, Leonards PEG, van Leeuwen SPJ, Hamers T.
2009. Competitive binding of poly- and perfluorinated compounds to the thyroid
hormone transport protein transthyretin. Toxicological Sciences 109:206-216.
Whitehead HD, Venier M, Wu Y, Eastman E, Urbanik S, Diamond ML, et al. 2021.
Fluorinated compounds in north american cosmetics. Environ Sci Tech Letters.
Xie W, Zhong W, Appenzeller BMR, Zhang J, Junaid M, Xu N. 2020. Nexus between
perfluoroalkyl compounds (PFCs) and human thyroid dysfunction: A systematic review
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compounds (PFOS/PFDA/PFOA) in juvenile chickens (gallus gallus). Arch Environ
Contam Toxicol 57:377-386.
Ylinen M, Auriola S. 1990. Tissue distribution and elimination of perfluorodecanoic acidin the rat after single intraperitoneal administration. Pharmacol Toxicol 66:45-48.
Zhang T, Sun H, Lin Y, Qin X, Zhang Y, Geng X, et al. 2013. Distribution of poly- andperfluoroalkyl substances in matched samples from pregnant women and carbon chain
length related maternal transfer. Environ Sci Technol 47:7974-7981.
Zhang W, Sheng N, Wang M, Zhang H, Dai J. 2016. Zebrafish reproductive toxicity
induced by chronic perfluorononanoate exposure. Aquat Toxicol 175:269-276.
Zhang Y, Beesoon S, Zhu L, Martin JW. 2013. Biomonitoring of perfluoroalkyl acids inhuman urine and estimates of biological half-life. Environ Sci Tech 47:10619-10627.
Zhao W, Cui R, Wang J, Dai J. 2017. Inhibition effects of perfluoroalkyl acids onprogesterone production in mltc-1. Journal of Environmental Sciences 56:272-280.
Zhou Y, Hu LW, Qian ZM, Chang JJ, King C, Paul G, et al. 2016. Association of
perfluoroalkyl substances exposure with reproductive hormone levels in adolescents: By
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APPENDIX A. LITERATURE SEARCH APPROACH ON THE MALE
REPRODUCTIVE TOXICITY OF PFNA AND ITS SALTS AND PFDA ANDITS SALTS
Searches of the published scientific literature on the developmental and reproductive
toxicity (DART) of PFNA and PFDA (and their salts) were conducted in February 2021.
The search sought to identify peer-reviewed open source and proprietary journal
articles, print and digital books, reports and gray literature that potentially reported
relevant toxicological and epidemiological information on the developmental andreproductive toxicity of these chemicals.
Three types of searches were conducted:•••

Primary searches in major biomedical databases, conducted by OEHHA librarian
Nancy Firchow, MLS
Searches in other data sources, including authoritative reviews and reports, anddatabases or web resources, conducted by OEHHA scientists
Additional focused searches, conducted by OEHHA scientists

Search process
The US Environmental Protection Agency (US EPA) Computational Toxicology(CompTox) Chemicals Dashboard (https://comptox.epa.gov/dashboard) was used to
identify synonyms for PFNA and PFDA (and their salts). The PubMed MeSH database(https://www.ncbi.nlm.nih.gov/mesh/) was used to identify subject headings and otherindex terms related to the chemicals, reproduction and development, and adverse
effects on reproduction and development.
Preliminary searches were conducted and results evaluated to identify additional
relevant search terms. The resulting search strategies were then executed in PubMed
twice for each chemical (and its salts), limiting the first search to human studies, and thesecond search to non-human studies. There were no restrictions in the searches on
exposure route or duration of exposure, or on publication language. The PubMed
search strategies (see Table A.1 and Table A. 2 below) were then tailored for use in the
additional databases and data sources listed below, according to the search interface
and features unique to each resource. For instance, MeSH terms were replaced with
Emtree terms for the Embase search strategies.
Data sources:
The following is a list of the major data sources (biomedical literature databases)
searched to find information on PFNA and its salts and PFDA and its salts.
•

PubMed (National Library of Medicine) (https://www.ncbi.nlm.nih.gov/pubmed)

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•••

Embase (https://www.embase.com)
Scopus (https://www.scopus.com)
SciFinder-n (https://scifinder-n.cas.org/)

In addition to the systematic literature searches, OEHHA asked the public to identify
pertinent references through a data call-in that was open from March 26, 2021 to May
10, 2021. No references were submitted for consideration.
Literature screening process
The results of these literature searches were uploaded to EndNote libraries (human andnon-human [i.e., experimental animal and cell-free] results were kept separate) andduplicates were removed. A total of 652 and 472 references were identified for PFNA
and its salts and PFDA and its salts respectively, through this initial literature search
process. Among these references, 44 were human or animal studies with information
directly relevant to male reproductive toxicity. In addition to the studies identified
through this initial, primary search process, other relevant studies were identified fromcitations in individual articles, through alert services (e.g., ScienceDirect, Google
Scholar, etc.), and through additional focused searches conducted as needed onmechanistic topics (e.g., role of thyroid system in male reproduction) and other areas(e.g., pharmacokinetics).
Use of Health Assessment Workspace Collaborative
The Health Assessment Workspace Collaborative (HAWC)(https://hawcproject.org/about/) was used as a tool in the systematic review of theliterature on the male reproductive toxicity of PFNA and PFDA and their salts, which
was a subset of the overall DART literature retrieved.
In using HAWC, the following steps were followed:• Importing the EndNote libraries into HAWC
The citations identified from all literature searches described above for each of the two
HAWC projects, (i.e., PFNA and its salts, PFDA and its salts) were uploaded into twocorresponding EndNote libraries, and duplicates were removed. Next, the EndNote
libraries were imported to HAWC for multi-level screening.
•

Defining specific inclusion and exclusion criteria for multi-level literature tags in
two HAWC projects.

•

Screening and tagging references

In Level 1 screening, the citations were screened independently by OEHHA scientists,based solely on titles and abstracts, to eliminate studies or articles that do not contain
information on PFNA and its salts, PFDA and its salts or on any of the key topics such
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as studies related to DART in humans and animals, pharmacokinetics, or relevantmechanisms. The initial screen was intended to identify all studies deemed to have areasonable possibility of containing information that could be useful for the review
process. During the screening of each HAWC project, citations were added to the otherproject when appropriate. Papers identified for inclusion during Level 1 screening were
tagged in HAWC according to key topics. A paper can be assigned (or tagged) to one
or more of the key topic(s). Citations that were identified as being relevant to male
reproductive toxicity during Level 1 sceening were moved to the second level ofscreening.
In Level 2 screening, the full papers were obtained for all citations that passed the Level1 screen and were identified as being relevant to male reproductive toxicity. These full
papers were screened independently by one OEHHA scientist, using similar
inclusion/exclusion criteria as was used in the Level 1 screening. However, Level 2
reviewers could make more accurate judgments about the relevance of the studies
because they were reviewing the full text of the articles, in addition to the title andabstract. Following Level 2 screening, the tagging of articles according to key topics
was updated.
Level 1 and 2 screenings were repeated as search results were updated, and withadditional relevant studies identified from citations in individual articles and alert
services (e.g., ScienceDirect, Google Scholar).
The basic search structure used for each of the searches is presented below, in Tables
A.1 and A.2. Similar search terms were used in searches of the four biomedical
literature databases. The search terms used for the PubMed searches are presented inTable A.1 (Human DART Study Searches) and Table A.2 (Animal DART Study
Searches). The same search strategies were used for PFNA and its salts and PFDA
and its salts. Only the group of terms used to identify the parent chemical was changed(Set #1 in each search). All searches were conducted in early February 2021.
Literature searches were last updated in July 2021. One hundred fourteen referenceswere cited in this document.

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Table A. 1 Human DART Study Searches
PubMed Search
Set#

Search Terms

Notes

Set
#

Search Terms

Notes

1

"Perfluorononanoic acid"[nm] OR"Perfluorononanoic acid"[tiab] OR"Heptadecafluorononanoic acid"[tiab] OR "Nonanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9heptadecafluoro- "[tiab] OR"PFNA "[tiab] OR "Nonanoic acid,heptadecafluoro- "[tiab] OR"2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9Heptadecafluorononanoic acid"[tiab] OR "C 1800 "[tiab] OR"Perfluoropelargonic acid "[tiab]
OR "perfluorononan-1-oic acid"[tiab] OR "Ammonium
perfluorononanoate "[tiab] OR"Ammonium perfluoropelargonate"[tiab] OR "APFN"[tiab] OR"APFNA"[tiab] OR"perfluorononanoate"[tiab] OR"Heptadecafluorononanoate"[tiab]
OR "Sodium
heptadecafluorononanoate"[tiab]
OR "sodium
perfluorononanoate"[tiab] OR"potassium
perfluorononanoate"[tiab] OR"potassium
heptadecafluorononanoate"[tiab]
OR 375-95-1[rn] OR 4149-604[rn] OR 21049-39-8[rn] OR
21049-38-7[rn]

PFNA
Terms

1

"perfluorodecanoic acid"[nm] OR"Perfluorodecanoic acid "[tiab] OR"Nonadecafluorodecanoic acid"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-"[tiab] OR"Nonadecafluorodecanoic acid"[tiab] OR"Decanoic acid, nonadecafluoro-"[tiab]
OR"2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10Nonadecafluorodecanoic acid"[tiab] OR"Perfluoro-1-nonanecarboxylic
acid"[tiab] OR "Perfluorocapric
acid"[tiab] OR "PFDA"[tiab] OR"nonadecafluorodecanoic acid"[tiab] OR"Nonadecafluoro-n-decanoic acid"[tiab]
OR "Perfluoro-n-decanoic acid"[tiab] OR"Ammonium
nonadecafluorodecanoate"[tiab] OR"perfluorodecanoate"[tiab] OR"nonadecafluorodecanoate"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-, ammonium salt"[tiab]
OR "Methyl Perfluorodecanoate"[tiab]
OR "Methyl
nonadecafluorodecanoate"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-, methyl ester "[tiab] OR"methylperfluorodecanoate"[tiab] OR
335-76-2[rn] OR 3108-42-7[rn] OR 30779-9[rn]

PFDA
Terms

Set
#

Search Terms

Notes

2

dart[sb]

PubMed DART
Subset3"abortion, spontaneous"[mh] OR "abortion*"[tiab] OR "Acrosome"[mh] OR"Acrosome"[tiab] OR "Adrenarche"[tiab] OR "androgen antagonists"[mh] OR"androgen*"[tiab] OR "androgens"[mh] OR "Androstenedione"[tiab] OR

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Additional DART
Terms

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Set#

Search Terms

Notes"anogenital distance"[tiab] OR "ano genital distance"[tiab] OR "anovulat*"[tiab]
OR "Aspermia"[tiab] OR "atretic follicle*"[tiab] OR "Azoospermia"[tiab] OR"birth defect*"[tiab] OR "birth weight"[mh] OR "birth weight"[tiab] OR "breast
feed*"[tiab] OR "breast feeding"[mh] OR "breastfeed*"[tiab] OR "chorionicvilli"[tiab] OR "conception*"[tiab] OR "congenital abnormalities"[mh] OR"Congenital"[tiab] OR "corpus luteum"[tiab] OR "cumulus cell*"[tiab] OR"cytotrophoblast*"[tiab] OR "decidua"[tiab] OR "deciduum"[tiab] OR "dnadamage"[mh] OR "ductus deferens"[tiab] OR "efferent duct*"[tiab] OR"ejaculat*"[tiab] OR "Embryo"[tiab] OR "Embryoes"[tiab] OR "embryonic andfetal development"[mh] OR "embryonic structures"[mh] OR "Embryonic"[tiab]
OR "embryotoxic*"[tiab] OR "endometri*"[tiab] OR "Epididymis"[mh] OR"Epididymis"[tiab] OR "erecti*"[tiab] OR "Estradiol"[tiab] OR "estrogenantagonists"[mh] OR "estrogen receptor modulators"[mh] OR "estrogen*"[tiab]
OR "estrogens"[mh] OR "Estrus"[tiab] OR "fallopian tube*"[tiab] OR "fallopian
tubes"[mh] OR "fecund*"[tiab] OR "Fertility"[mh] OR "Fertility"[tiab] OR"Fertilization"[tiab] OR "Fetal"[tiab] OR "Fetus"[mh] OR "Fetus"[tiab] OR"foetal"[tiab] OR "foetus"[tiab] OR "follicle stimulating hormone"[tiab] OR"FSH"[tiab] OR "genetic diseases, inborn"[mh] OR "genital diseases,
female"[mh] OR "genital diseases, male"[mh] OR "genital*"[tiab] OR"genitalia"[mh] OR "germ cell*"[tiab] OR "germ cells"[mh] OR "gestat*"[tiab] OR"gonad*"[tiab] OR "gonadal disorders"[mh] OR "gonadal hormones"[mh] OR"gonadotropins"[mh] OR "gonads"[mh] OR "graafian follicle*"[tiab] OR"granulosa cell*"[tiab] OR "human development"[mh] OR "Implantation"[tiab]
OR "in utero"[tiab] OR "infant*"[tiab] OR "infant, newborn"[mh] OR"infertil*"[tiab] OR "Inhibin"[tiab] OR "Intrauterine"[tiab] OR "Lactation"[tiab] OR"lactation disorders"[mh] OR "leydig cell*"[tiab] OR "leydig cells"[mh] OR"LH"[tiab] OR "luteal cell*"[tiab] OR "luteinizing hormone"[tiab] OR "maternal
exposure"[mh] OR "Maternal"[tiab] OR "Menses"[tiab] OR "menstrua*"[tiab] OR"miscarriage*"[tiab] OR "neonat*"[tiab] OR "Oligospermia"[tiab] OR"oocyte*"[tiab] OR "Oogonia"[tiab] OR "Ova"[tiab] OR "ovarian follicle*"[tiab]
OR "Ovarian"[tiab] OR "Ovaries"[tiab] OR "Ovary"[mh] OR "Ovary"[tiab] OR"oviduct*"[tiab] OR "oviducts"[mh] OR "ovulat*"[tiab] OR "Ovum"[mh] OR"Ovum"[tiab] OR "paternal exposure"[mh] OR "Paternal"[tiab] OR"peripubert*"[tiab] OR "pituitary hormones"[mh] OR "placenta*"[tiab] OR"placenta"[mh] OR "placental hormones"[mh] OR "preconception*"[tiab] OR"pre conception*"[tiab] OR "pregnan*"[tiab] OR "pregnancy complications"[mh]OR "pregnancy"[mh] OR "prenatal exposure delayed effects"[mh] OR"prenatal"[tiab] OR "Pre-natal"[tiab] OR "Preterm"[tiab] OR "Pre-term"[tiab] OR"primary follicle*"[tiab] OR "Progesterone"[tiab] OR "progestin*"[tiab] OR"progestins"[mh] OR "Prostate"[mh] OR "Prostate"[tiab] OR "reproduct*"[tiab]
OR "reproductive physiological phenomena"[mh] OR "secondary follicle*"[tiab]
OR "Semen"[mh] OR "Semen"[tiab] OR "seminal vesicle*"[tiab] OR "seminal
vesicles"[mh] OR "Seminal"[tiab] OR "seminiferous epithelium"[tiab] OR"seminiferous tubule*"[tiab] OR "seminiferous tubules"[mh] OR"Seminiferous"[tiab] OR "sertoli cells"[mh] OR "sertoli cell*"[tiab] OR "sexual
development"[mh] OR "Sperm"[tiab] OR "spermatid*"[tiab] OR

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Set#

Search Terms

Notes"spermatocyte*"[tiab] OR "spermatogenesis"[tiab] OR "Spermatogonia"[tiab]
OR "Spermatozoa"[mh] OR "Spermatozoa"[tiab] OR "Sterile"[tiab] OR"Sterility"[tiab] OR "stillbirth*"[tiab] OR "Stillborn"[tiab] OR"syncytiotrophoblast*"[tiab] OR "teratogen*"[tiab] OR "teratogens"[mh] OR"tertiary follicle*"[tiab] OR "Testes"[tiab] OR "testic*"[tiab] OR "Testis"[mh] OR"Testis"[tiab] OR "Testosterone"[tiab] OR "theca cell*"[tiab] OR "thyroid
hormones"[mh] OR "trophoblast*"[tiab] OR "urogenital abnormalities"[mh] OR"urogenital*"[tiab] OR "Uterine"[tiab] OR "Uterus"[mh] OR "Uterus"[tiab] OR"vagina*"[tiab] OR "vas deferens"[mh] OR "vas deferens"[tiab] OR"zygote*"[tiab]
4"spermatozoa"[mh] OR "acrosome reaction"[mh] OR "Sperm Capacitation"[mh]OR "sperm transport"[mh] OR "sperm-ovum interactions"[mh] OR"acrosome"[tiab] OR "spermatozoa"[tiab] OR "sperm"[tiab] OR"spermatogonia"[tiab] OR "spermatophore*"[tiab] OR "spermatocyte*"[tiab] OR"spermatid*"[tiab] OR "spermatogenesis"[tiab] OR "capacitation"[tiab] OR(("Germ cells"[mh] OR "germ cell*"[tiab]) AND ("male"[mh] OR "male"[tiab]))OR "Leydig cells"[mh] OR "leydig cell*"[tiab] OR "sertoli cells"[mh] OR "sertoli
cell*"[tiab] OR "cytoskeleton"[tiab] OR "gonadal somatic cells"[tiab] OR "folliclestimulating hormone"[mh] OR "testosterone congeners"[mh] OR "luteinizing
hormone"[mh] OR "androgens"[mh] OR "follicle stimulating hormone*"[tiab] OR"FSH"[tiab] OR "luteinizing hormone"[tiab] OR "LH"[tiab] OR "Inhibin"[tiab] OR"testosterone"[tiab] OR "prolactin"[tiab] OR "androgen*"[tiab] OR "gonadotropin
releasing hormone"[tiab] OR "GnRH"[tiab] OR "steroidogenic"[tiab] OR"reproductive hormone*"[tiab] OR "reproductive steroid hormone*"[tiab] OR"sex steroid*"[tiab] OR "hypothalamic pituitary gonadal axis"[tiab] OR "hpgaxis"[tiab] OR "hypothalamic pituitary adrenal axis"[tiab] OR "hypothalamic
pituitary thyroid axis"[tiab] OR "CYP3A4"[tiab] OR "CYP17A1"[tiab] OR"aromatase"[tiab] OR "receptors, gonadotropin"[mh] OR "receptors, pituitaryhormone"[mh] OR "receptors, estrogen"[mh] OR "receptors, oxytocin"[mh] OR"receptors, androgen"[mh] OR "hormone receptor*"[tiab] OR "lh receptor*"[tiab]
OR "gonadotropin receptor*"[tiab] OR "estrogen receptor*"[tiab] OR "oestrogen
receptor*"[tiab] OR "fsh receptor*"[tiab] OR "androgen receptor*"[tiab] OR"testosterone receptor*"[tiab] OR "prolactin receptor*"[tiab] OR "DNA
Adducts"[mh] OR "comet assay"[mh] OR "Germ-line mutation"[mh] OR"Mutagenesis"[mh] OR "Mutagenicity tests"[mh] OR "sister chromatid
exchange"[mh] OR "Mutation"[mh] OR "DNA Repair"[mh] OR "genomic
instability"[mh] OR "Aneuploidy"[mh] OR "ames assay"[tiab] OR "amestest"[tiab] OR "bacterial reverse mutation assay"[tiab] OR "clastogen*"[tiab] OR"genetic toxicology"[tiab] OR "hyperploid"[tiab] OR "micronucleus test"[tiab] OR"tetraploid"[tiab] OR "chromosome aberrations"[tiab] OR "mutation*"[tiab] OR"chromosome translocation*"[tiab] OR "dna protein crosslink*"[tiab] OR "dnadamag*"[tiab] OR "dna inhibit*"[tiab] OR "Micronuclei"[tiab] OR"Micronucleus"[tiab] OR "Mutagens"[tiab] OR "strand break*"[tiab] OR"unscheduled dna synthes*"[tiab] OR "chromosomal aberration*"[tiab] OR"chromosome aberration"[tiab] OR "chromosomal abnormalit*"[tiab] OR

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Characteristics ofMale
Reproductive
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Set#

Search Terms

Notes"chromosome abnormalit*"[tiab] OR "chromosome damage*"[tiab] OR"genotoxic*"[tiab] OR "adduct formation"[tiab] OR "dna adduct*"[tiab] OR "dnabreak*"[tiab] OR "dsdna break*"[tiab] OR ("DNA"[tiab]AND "Crosslink"[tiab])
OR "microsatellite-instability"[tiab] OR "chromosomal-instability"[tiab] OR"binucleation"[tiab] OR "binucleated"[tiab] OR (("comet assay"[tiab] OR"Mutagenic"[tiab] OR "Mutagenicity"[tiab] OR "mutations"[tiab] OR"chromosomal-aberration-test"[tiab] OR "sister chromatid exchange"[tiab] OR"SOS-response"[tiab] OR "polyploid*"[tiab] OR "genomic instability"[tiab] OR"dna repair*"[tiab] OR "aneuploid*"[tiab]) NOT "Medline"[Filter]) OR"epigenesis, genetic"[mh] OR "epigenomics"[mh] OR "DNA methylation"[mh]OR "gene silencing"[mh] OR "histone deacetylases"[mh] OR "RNAInterference"[mh] OR "microRNAs"[mh] OR "rna, small interfering"[mh] OR"cpg islands/genetics"[mh] OR "cpg island methylator"[tiab] OR "cpg island
methylation"[tiab] OR "epigenotype"[tiab] OR "epimutation*"[tiab] OR"methylation associated silencing"[tiab] OR "histone tail modifications"[tiab] OR"histone tail modification"[tiab] OR "chromatin organization"[tiab] OR"chromatin packag*"[tiab] OR "histone modification"[tiab] OR "histone
retention"[tiab] OR "epigenetic*"[tiab] OR "epigenomic*"[tiab] OR "RNAInterference"[tiab] OR "noncoding rna"[tiab] OR "ncRNA"[tiab] OR "geneactivation"[tiab] OR "proteasome"[tiab] OR "DNA methylation"[tiab] OR"Methylation"[Title] OR "CIMP"[tiab] OR "Free Radicals"[mh] OR "Reactive
Oxygen Species"[mh] OR "Oxidative stress"[mh] OR "Electron Transport"[mh]OR "free radical*"[tiab] OR "oxygen radical*"[tiab] OR "Oxidative stress"[tiab]
OR "redox balance"[tiab] OR "oxidative damage*"[tiab] OR "Reactive Oxygen
Species"[tiab] OR "reactive nitrogen species"[tiab] OR "superoxide
radical*"[tiab] OR "hydroxyl radical"[tiab] OR "glutathione deplet*"[tiab] OR"oxidative protein damage"[tiab] OR "C-reactive protein"[mh] OR"eosinophils"[mh] OR ("fibrinogen"[tiab]AND "Inflammation"[tiab]) OR"chronicinflammation"[tiab] OR "chronically inflamed"[tiab] OR "acuteinflammat*"[tiab] OR "infiltrating leukocyt*"[tiab] OR "inflammatoryleukocyte"[tiab] OR "inflammatory-leukocytes"[tiab] OR "leukocyte
infiltrat*"[tiab] OR "pro-inflammatory"[tiab] OR "proinflammatory"[tiab] OR"macrophage-recruitment"[tiab] OR "macrophage inflammatory proteins"[tiab]
OR "macrophage colony stimulating factor*"[tiab] OR "urethritis"[tiab] OR"prostatitis"[tiab] OR "seminal vesiculitis"[tiab] OR "epididymitis"[tiab] OR"orchitis"[tiab]
5("hypothalamic pituitary ovarian axis"[tiab] OR "hpo axis"[tiab] OR "gonadal
hormones"[mh] OR "pituitary hormones"[mh] OR "gonadotropin releasing
hormone"[mh] OR "follicle stimulating hormone"[mh] OR "testosterone"[mh]OR "receptors, gonadotropin"[mh] OR "receptors, pituitary hormone"[mh] OR"receptors, estrogen"[mh] OR "receptors, oxytocin"[mh] OR "estrogens"[mh]OR "estradiol"[mh] OR "estriol"[mh] OR "estrone"[mh] OR "luteinizing
hormone"[mh] OR "androgens"[mh] OR "hydroxyprogesterones"[mh] OR"Dehydroepiandrosterone"[mh] OR "Androstenedione"[mh] OR"Androstenediol"[mh] OR "Dihydrotestosterone"[mh] OR "androgen

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Key
Characteristics ofFemaleReproductive
Toxicity

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Set#

Search Terms

Notes

receptor*"[tiab] OR "estradiol receptor*"[tiab] OR "estrogen receptor*"[tiab] OR"follicle stimulating hormone"[tiab] OR "fsh receptor*"[tiab] OR "gonadotropin
releasing hormone"[tiab] OR "hormone receptor*"[tiab] OR "lh receptor*"[tiab]
OR "luteinizing hormone"[tiab] OR "oestrogen receptor*"[tiab] OR "ovarian
hormone*"[tiab] OR "ovarian steroid*"[tiab] OR "oxytocin receptor*"[tiab] OR"plasma membrane receptor*"[tiab] OR "prolactin receptor*"[tiab] OR"reproductive hormone*"[tiab] OR "sex hormone*"[tiab] OR "testosterone
receptor*"[tiab] OR "activin"[tiab] OR "estradiol"[tiab] OR "estriol"[tiab] OR"estrogen"[tiab] OR "estrone"[tiab] OR "FSH"[tiab] OR "gnrh"[tiab] OR"gonadotropin*"[tiab] OR "gonadotropin receptor*"[tiab] OR "hcg"[tiab] OR"inhibin"[tiab] OR "LH"[tiab] OR "LHRH"[tiab] OR "oestriol"[tiab] OR"oestradiol"[tiab] OR "oestrogen"[tiab] OR "oestrone"[tiab] OR "Oxytocin"[tiab]
OR "progesterone"[tiab] OR "prolactin"[tiab] OR "Steroidogenic"[tiab] OR"testosterone"[tiab] OR "Pregnenolone"[tiab] OR "17alpha hydroxy 6
methylene progesterone"[Supplementary Concept] OR"Dehydroepiandrosterone"[tiab] OR "DHEA"[tiab] OR "DHEAS"[tiab] OR"Androstenedione"[tiab] OR "Androstenediol"[tiab] OR"Dihydrotestosterone"[tiab] OR "steroidogenic acute regulatory
protein"[Supplementary Concept] OR "steroidogenic acute regulatory
protein"[tiab] OR "star protein"[tiab] OR "cholesterol side chain cleavage
enzyme"[mh] OR "cholesterol side chain cleavage enzyme"[tiab] OR"cholesterol desmolase"[tiab] OR "cytochrome p 450 scc"[tiab] OR"P450scc"[tiab] OR "CYP11A"[tiab] OR "CYP11A1"[tiab] OR "17alphahydroxylase"[tiab] OR "17,20 lyase"[tiab] OR "P450c17"[tiab] OR "CYP17"[tiab]
OR "aromatase"[mh] OR "aromatase"[tiab] OR "cytochrome p450 family
19"[mh] OR "cytochrome p450 family 19"[tiab] OR "P450arom"[tiab] OR"CYP19"[tiab] OR "3 or 17 beta hydroxysteroid
dehydrogenase"[Supplementary Concept] OR "3beta hydroxysteroid
dehydrogenase"[tiab] OR "3beta hsd"[tiab] OR "17beta hydroxysteroid
dehydrogenase"[tiab] OR "17beta hsd*"[tiab] OR "5alpha-reductase"[tiab] OR("DNA Adducts"[mh] OR "Comet Assay"[mh] OR "Germ-line mutation"[mh] OR"Mutagenesis"[mh] OR "Mutagenicity tests"[mh] OR "Sister-chromatid
exchange"[mh] OR "Mutation"[mh] OR "Ames-Assay"[tiab] OR "Amestest"[tiab] OR "Bacterial-Reverse-Mutation-Assay"[tiab] OR "clastogen*"[tiab]
OR "dna repair*"[tiab] OR "Genetic-toxicology"[tiab] OR "hyperploid"[tiab] OR"micronucleus-test"[tiab] OR "tetraploid"[tiab] OR "Chromosomeaberrations"[tiab] OR "DNA-damage"[tiab] OR "mutation*"[tiab] OR"chromosome-translocations"[tiab] OR "dna protein crosslink*"[tiab] OR "dnadamag*"[tiab] OR "dna inhibit*"[tiab] OR "Micronuclei"[tiab] OR"Micronucleus"[tiab] OR "Mutagens"[tiab] OR "strand break*"[tiab] OR"unscheduled dna synthes*"[tiab] OR "chromosomal aberration*"[tiab] OR"chromosome aberration*"[tiab] OR "chromosomal abnormalit*"[tiab] OR"chromosome abnormalit*"[tiab] OR "genotoxic*"[tiab] OR "adductformation"[tiab] OR "dna adduct*"[tiab] OR "dna break*"[tiab] OR "dsdna
break*"[tiab]) OR ("epigenesis, genetic"[mh] OR "epigenomics"[mh] OR "DNAmethylation"[mh] OR "gene silencing"[mh] OR "histone deacetylases"[mh] OR

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Search Terms

Notes"RNA Interference"[mh] OR "microRNAs"[mh] OR "rna, small interfering"[mh]OR "cpg islands/genetics"[mh] OR "cpg island methylator"[tiab] OR "cpg island
methylation"[tiab] OR "epigenotype"[tiab] OR "epimutation*"[tiab] OR"methylation associated silencing"[tiab] OR "histone tail modifications"[tiab] OR"histone tail modification"[tiab] OR "chromatin organization"[tiab] OR"chromatin packag*"[tiab] OR "histone modification"[tiab] OR "histone
retention"[tiab] OR "epigenetic*"[tiab] OR "epigenomic*"[tiab] OR "RNAInterference"[tiab] OR "noncoding rna"[tiab] OR "ncRNA"[tiab] OR "geneactivation"[tiab] OR "proteasome"[tiab] OR "DNA methylation"[tiab] OR"Methylation"[Title] OR "CIMP"[tiab]) OR ("mitochondria"[mh] OR "oxidative
phosphorylation"[mh] OR "mitochrondria*"[tiab] OR "oxidative
phosphorylation"[tiab] OR "oxidative damage"[tiab] OR "fatty acid betaoxidation"[tiab] OR "calcium buffering"[tiab] OR "ca2 buffering"[tiab] OR"mitochondrial dna mutation*"[tiab] OR "mtdna mutation*"[tiab] OR"mtDNA"[tiab]) OR ("Free Radicals"[mh] OR "Reactive Oxygen Species"[mh]OR "Oxidative stress"[mh] OR "Electron Transport"[mh] OR "free radical*"[tiab]
OR "oxygen radical*"[tiab] OR "Oxidative stress"[tiab] OR "redox balance"[tiab]
OR "oxidative damage*"[tiab] OR "Reactive Oxygen Species"[tiab] OR"reactive nitrogen species"[tiab] OR "superoxide radical*"[tiab] OR "hydroxyl
radical"[tiab] OR "glutathione deplet*"[tiab] OR "oxidative protein
damage"[tiab]) OR ("cytotoxicity, immunologic"[mh] OR "Immunologic
Factors"[mh] OR "Immunomodulation"[mh] OR "B-Cell Activation Factor
Receptor"[mh] OR "Antigenic Modulation"[mh] OR "B-Cell Activating
Factor"[mh] OR "Immunologic Factors"[Pharmacological Action] OR "b-cellactivation"[tiab] OR "immune surveillance"[tiab] OR "immune suppress*"[tiab]
OR "immunostimulant"[tiab] OR "immune-activation"[tiab] OR"immunodeficien*"[tiab] OR "somatic-hypermutation"[tiab] OR "immuneactivation"[tiab] OR "immune-system-activation"[tiab] OR "Chronic-antigenicstimulation"[tiab] OR "immunosuppress*"[tiab] OR "immune
dysregulation"[tiab]) OR ("signal transduction"[mh] OR "signaltransduction"[tiab] OR "signal pathway*"[tiab] OR "signaling pathway*"[tiab]
OR "ion channel"[tiab] OR "signaling system*"[tiab] OR "cell signal*"[tiab] OR"cellular signal*"[tiab] OR "intracellular signal*"[tiab] OR "signal cascade*"[tiab]
OR "signaling cascade*"[tiab] OR "second messenger*"[tiab] OR "calcium
signal*"[tiab]) OR ("cell communication"[mh] OR "gap junctions"[mh] OR"connexins"[mh] OR "connexins"[Supplementary Concept] OR "cellcommunication*"[tiab] OR "cellular communication*"[tiab] OR "intracellularcommunication*"[tiab] OR "cell interaction*"[tiab] OR "gap junction*"[tiab] OR"connexin*"[tiab]) OR ("Apoptosis"[mh] OR "cytotoxicity, immunologic"[mh] OR"Caspases"[mh] OR "autophagy"[mh] OR "necrosis"[mh] OR "Autolysis"[mh]OR "Angiogenesis Modulating Agents"[mh] OR "Angiogenesis Inducing
Agents"[Pharmacological Action] OR "Angiogenesis Inducing Agents"[mh] OR"neovascularization, pathologic"[mh] OR "Cell Proliferation"[mh] OR"homeostasis"[mh] OR "Cyclin-Dependent Kinases"[mh] OR "CyclinDependent Kinase Inhibitor Proteins"[mh] OR "Mitogens"[mh] OR"Mitogens"[Pharmacological Action] OR "cell hypoxia"[mh] OR

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Search Terms

Notes"angiogenic"[tiab] OR "Apoptosis"[tiab] OR "autophagy"[tiab] OR"Caspases"[tiab] OR "cell cycle control*"[tiab] OR "cell cycle arrest"[tiab] OR"cell hypoxia"[tiab] OR "Cell Proliferation"[tiab] OR "cellular-energetics"[tiab]
OR "cellular-hypoxia"[tiab] OR "cellular proliferation"[tiab] OR "cellular
replication*"[tiab] OR "Cytogenesis"[tiab] OR "Cytogenic"[tiab] OR"Cytotoxin"[tiab] OR "hepatocellular-proliferation"[tiab] OR "hyperplasia"[tiab]
OR "hypoxic cell*"[tiab] OR "mitogenesis"[tiab] OR "mitotic checkpoint*"[tiab]
OR "Neoplasia"[tiab] OR "p53 delet*"[tiab] OR "p53 inactivat*"[tiab] OR "p53inhibit*"[tiab] OR "prb delet*"[tiab] OR "prb inactivat*"[tiab] OR "prbinhibit*"[tiab] OR "programmed cell death"[tiab] OR ("Rb"[All Fields] AND"p16ink4a inactiv*"[tiab]) OR "retinoblastoma-protein"[tiab] OR"senescence"[tiab] OR "senescent"[tiab] OR "survivin"[tiab]) OR"microtubules"[mh] OR "spindle apparatus"[mh] OR "microtubule organizing
center"[mh] OR "microtubule*"[tiab] OR "spindle formation"[tiab] OR "spindle
apparatus"[tiab] OR "meiotic spindle*"[tiab] OR "mitotic spindle*"[tiab])
6#2 OR #3 OR #4 OR #5

Combine DART
concept groups

7#1 AND #6

CombineChemical &DART

8#7 NOT (animals[mh] NOT humans[mh])Remove AnimalStudies - FINAL

Table A. 2 Animal DART Study Searches
PubMed Search
Set#

Search Terms

Notes

Set
#

Search Terms

Notes

1

"Perfluorononanoic acid"[nm] OR"Perfluorononanoic acid"[tiab] OR"Heptadecafluorononanoic acid"[tiab] OR "Nonanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9heptadecafluoro- "[tiab] OR"PFNA "[tiab] OR "Nonanoic acid,heptadecafluoro- "[tiab] OR"2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9Heptadecafluorononanoic acid"[tiab] OR "C 1800 "[tiab] OR"Perfluoropelargonic acid "[tiab]
OR "perfluorononan-1-oic acid

PFNA
Terms

1

"perfluorodecanoic acid"[nm] OR"Perfluorodecanoic acid "[tiab] OR"Nonadecafluorodecanoic acid"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-"[tiab] OR"Nonadecafluorodecanoic acid"[tiab] OR"Decanoic acid, nonadecafluoro-"[tiab]
OR"2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10Nonadecafluorodecanoic acid"[tiab] OR"Perfluoro-1-nonanecarboxylic
acid"[tiab] OR "Perfluorocapric

PFDA
Terms

PFNA and its salts andPFDA and its salts

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"[tiab] OR "Ammonium
perfluorononanoate "[tiab] OR"Ammonium perfluoropelargonate"[tiab] OR "APFN"[tiab] OR"APFNA"[tiab] OR"perfluorononanoate"[tiab] OR"Heptadecafluorononanoate"[tiab]
OR "Sodium
heptadecafluorononanoate"[tiab]
OR "sodium
perfluorononanoate"[tiab] OR"potassium
perfluorononanoate"[tiab] OR"potassium
heptadecafluorononanoate"[tiab]
OR 375-95-1[rn] OR 4149-604[rn] OR 21049-39-8[rn] OR
21049-38-7[rn]

Set
#
2
3

acid"[tiab] OR "PFDA"[tiab] OR"nonadecafluorodecanoic acid"[tiab] OR"Nonadecafluoro-n-decanoic acid"[tiab]
OR "Perfluoro-n-decanoic acid"[tiab] OR"Ammonium
nonadecafluorodecanoate"[tiab] OR"perfluorodecanoate"[tiab] OR"nonadecafluorodecanoate"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-, ammonium salt"[tiab]
OR "Methyl Perfluorodecanoate"[tiab]
OR "Methyl
nonadecafluorodecanoate"[tiab] OR"Decanoic acid,
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10nonadecafluoro-, methyl ester "[tiab] OR"methylperfluorodecanoate"[tiab] OR
335-76-2[rn] OR 3108-42-7[rn] OR 30779-9[rn]Search Terms

Notes

dart[sb]

PubMed DART
Subset"abortion, spontaneous"[mh] OR "abortion*"[tiab] OR "Acrosome"[mh] OR"Acrosome"[tiab] OR "Adrenarche"[tiab] OR "androgen antagonists"[mh] OR"androgen*"[tiab] OR "androgens"[mh] OR "Androstenedione"[tiab] OR"anogenital distance"[tiab] OR "ano genital distance"[tiab] OR "anovulat*"[tiab]
OR "Aspermia"[tiab] OR "atretic follicle*"[tiab] OR "Azoospermia"[tiab] OR"birth defect*"[tiab] OR "birth weight"[mh] OR "birth weight"[tiab] OR "breast
feed*"[tiab] OR "breast feeding"[mh] OR "breastfeed*"[tiab] OR "chorionicvilli"[tiab] OR "conception*"[tiab] OR "congenital abnormalities"[mh] OR"Congenital"[tiab] OR "corpus luteum"[tiab] OR "cumulus cell*"[tiab] OR"cytotrophoblast*"[tiab] OR "decidua"[tiab] OR "deciduum"[tiab] OR "dnadamage"[mh] OR "ductus deferens"[tiab] OR "efferent duct*"[tiab] OR"ejaculat*"[tiab] OR "Embryo"[tiab] OR "Embryoes"[tiab] OR "embryonic andfetal development"[mh] OR "embryonic structures"[mh] OR "Embryonic"[tiab]
OR "embryotoxic*"[tiab] OR "endometri*"[tiab] OR "Epididymis"[mh] OR"Epididymis"[tiab] OR "erecti*"[tiab] OR "Estradiol"[tiab] OR "estrogenantagonists"[mh] OR "estrogen receptor modulators"[mh] OR "estrogen*"[tiab]
OR "estrogens"[mh] OR "Estrus"[tiab] OR "fallopian tube*"[tiab] OR "fallopian
tubes"[mh] OR "fecund*"[tiab] OR "Fertility"[mh] OR "Fertility"[tiab] OR"Fertilization"[tiab] OR "Fetal"[tiab] OR "Fetus"[mh] OR "Fetus"[tiab] OR"foetal"[tiab] OR "foetus"[tiab] OR "follicle stimulating hormone"[tiab] OR"FSH"[tiab] OR "genetic diseases, inborn"[mh] OR "genital diseases,

Additional DARTTerms

PFNA and its salts andPFDA and its salts

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Set#

Search Terms

Notes

female"[mh] OR "genital diseases, male"[mh] OR "genital*"[tiab] OR"genitalia"[mh] OR "germ cell*"[tiab] OR "germ cells"[mh] OR "gestat*"[tiab]
OR "gonad*"[tiab] OR "gonadal disorders"[mh] OR "gonadal hormones"[mh]OR "gonadotropins"[mh] OR "gonads"[mh] OR "graafian follicle*"[tiab] OR"granulosa cell*"[tiab] OR "human development"[mh] OR "Implantation"[tiab]
OR "in utero"[tiab] OR "infant*"[tiab] OR "infant, newborn"[mh] OR"infertil*"[tiab] OR "Inhibin"[tiab] OR "Intrauterine"[tiab] OR "Lactation"[tiab]
OR "lactation disorders"[mh] OR "leydig cell*"[tiab] OR "leydig cells"[mh] OR"LH"[tiab] OR "luteal cell*"[tiab] OR "luteinizing hormone"[tiab] OR "maternal
exposure"[mh] OR "Maternal"[tiab] OR "Menses"[tiab] OR "menstrua*"[tiab]
OR "miscarriage*"[tiab] OR "neonat*"[tiab] OR "Oligospermia"[tiab] OR"oocyte*"[tiab] OR "Oogonia"[tiab] OR "Ova"[tiab] OR "ovarian follicle*"[tiab]
OR "Ovarian"[tiab] OR "Ovaries"[tiab] OR "Ovary"[mh] OR "Ovary"[tiab] OR"oviduct*"[tiab] OR "oviducts"[mh] OR "ovulat*"[tiab] OR "Ovum"[mh] OR"Ovum"[tiab] OR "paternal exposure"[mh] OR "Paternal"[tiab] OR"peripubert*"[tiab] OR "pituitary hormones"[mh] OR "placenta*"[tiab] OR"placenta"[mh] OR "placental hormones"[mh] OR "preconception*"[tiab] OR"pre conception*"[tiab] OR "pregnan*"[tiab] OR "pregnancy complications"[mh]OR "pregnancy"[mh] OR "prenatal exposure delayed effects"[mh] OR"prenatal"[tiab] OR "Pre-natal"[tiab] OR "Preterm"[tiab] OR "Pre-term"[tiab]
OR "primary follicle*"[tiab] OR "Progesterone"[tiab] OR "progestin*"[tiab] OR"progestins"[mh] OR "Prostate"[mh] OR "Prostate"[tiab] OR "reproduct*"[tiab]
OR "reproductive physiological phenomena"[mh] OR "secondary follicle*"[tiab]
OR "Semen"[mh] OR "Semen"[tiab] OR "seminal vesicle*"[tiab] OR "seminal
vesicles"[mh] OR "Seminal"[tiab] OR "seminiferous epithelium"[tiab] OR"seminiferous tubule*"[tiab] OR "seminiferous tubules"[mh] OR"Seminiferous"[tiab] OR "sertoli cells"[mh] OR "sertoli cell*"[tiab] OR "sexual
development"[mh] OR "Sperm"[tiab] OR "spermatid*"[tiab] OR"spermatocyte*"[tiab] OR "spermatogenesis"[tiab] OR "Spermatogonia"[tiab]
OR "Spermatozoa"[mh] OR "Spermatozoa"[tiab] OR "Sterile"[tiab] OR"Sterility"[tiab] OR "stillbirth*"[tiab] OR "Stillborn"[tiab] OR"syncytiotrophoblast*"[tiab] OR "teratogen*"[tiab] OR "teratogens"[mh] OR"tertiary follicle*"[tiab] OR "Testes"[tiab] OR "testic*"[tiab] OR "Testis"[mh] OR"Testis"[tiab] OR "Testosterone"[tiab] OR "theca cell*"[tiab] OR "thyroid
hormones"[mh] OR "trophoblast*"[tiab] OR "urogenital abnormalities"[mh] OR"urogenital*"[tiab] OR "Uterine"[tiab] OR "Uterus"[mh] OR "Uterus"[tiab] OR"vagina*"[tiab] OR "vas deferens"[mh] OR "vas deferens"[tiab] OR"zygote*"[tiab]

4"spermatozoa"[mh] OR "acrosome reaction"[mh] OR "Sperm
Capacitation"[mh] OR "sperm transport"[mh] OR "sperm-ovum
interactions"[mh] OR "acrosome"[tiab] OR "spermatozoa"[tiab] OR"sperm"[tiab] OR "spermatogonia"[tiab] OR "spermatophore*"[tiab] OR"spermatocyte*"[tiab] OR "spermatid*"[tiab] OR "spermatogenesis"[tiab] OR"capacitation"[tiab] OR (("Germ cells"[mh] OR "germ cell*"[tiab]) AND("male"[mh] OR "male"[tiab])) OR "Leydig cells"[mh] OR "leydig cell*"[tiab] OR

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Key
Characteristics ofMale
Reproductive
Toxicity

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Set#

Search Terms

Notes"sertoli cells"[mh] OR "sertoli cell*"[tiab] OR "cytoskeleton"[tiab] OR "gonadal
somatic cells"[tiab] OR "follicle-stimulating hormone"[mh] OR "testosterone
congeners"[mh] OR "luteinizing hormone"[mh] OR "androgens"[mh] OR"follicle stimulating hormone*"[tiab] OR "FSH"[tiab] OR "luteinizing
hormone"[tiab] OR "LH"[tiab] OR "Inhibin"[tiab] OR "testosterone"[tiab] OR"prolactin"[tiab] OR "androgen*"[tiab] OR "gonadotropin releasing
hormone"[tiab] OR "GnRH"[tiab] OR "steroidogenic"[tiab] OR "reproductive
hormone*"[tiab] OR "reproductive steroid hormone*"[tiab] OR "sexsteroid*"[tiab] OR "hypothalamic pituitary gonadal axis"[tiab] OR "hpgaxis"[tiab] OR "hypothalamic pituitary adrenal axis"[tiab] OR "hypothalamic
pituitary thyroid axis"[tiab] OR "CYP3A4"[tiab] OR "CYP17A1"[tiab] OR"aromatase"[tiab] OR "receptors, gonadotropin"[mh] OR "receptors, pituitaryhormone"[mh] OR "receptors, estrogen"[mh] OR "receptors, oxytocin"[mh] OR"receptors, androgen"[mh] OR "hormone receptor*"[tiab] OR "lhreceptor*"[tiab] OR "gonadotropin receptor*"[tiab] OR "estrogen
receptor*"[tiab] OR "oestrogen receptor*"[tiab] OR "fsh receptor*"[tiab] OR"androgen receptor*"[tiab] OR "testosterone receptor*"[tiab] OR "prolactin
receptor*"[tiab] OR "DNA Adducts"[mh] OR "comet assay"[mh] OR "Germ-line
mutation"[mh] OR "Mutagenesis"[mh] OR "Mutagenicity tests"[mh] OR "sister
chromatid exchange"[mh] OR "Mutation"[mh] OR "DNA Repair"[mh] OR"genomic instability"[mh] OR "Aneuploidy"[mh] OR "ames assay"[tiab] OR"ames test"[tiab] OR "bacterial reverse mutation assay"[tiab] OR"clastogen*"[tiab] OR "genetic toxicology"[tiab] OR "hyperploid"[tiab] OR"micronucleus test"[tiab] OR "tetraploid"[tiab] OR "chromosome
aberrations"[tiab] OR "mutation*"[tiab] OR "chromosome translocation*"[tiab]
OR "dna protein crosslink*"[tiab] OR "dna damag*"[tiab] OR "dna inhibit*"[tiab]
OR "Micronuclei"[tiab] OR "Micronucleus"[tiab] OR "Mutagens"[tiab] OR"strand break*"[tiab] OR "unscheduled dna synthes*"[tiab] OR "chromosomal
aberration*"[tiab] OR "chromosome aberration"[tiab] OR "chromosomal
abnormalit*"[tiab] OR "chromosome abnormalit*"[tiab] OR "chromosome
damage*"[tiab] OR "genotoxic*"[tiab] OR "adduct formation"[tiab] OR "dnaadduct*"[tiab] OR "dna break*"[tiab] OR "dsdna break*"[tiab] OR("DNA"[tiab]AND "Crosslink"[tiab]) OR "microsatellite-instability"[tiab] OR"chromosomal-instability"[tiab] OR "binucleation"[tiab] OR "binucleated"[tiab]
OR (("comet assay"[tiab] OR "Mutagenic"[tiab] OR "Mutagenicity"[tiab] OR"mutations"[tiab] OR "chromosomal-aberration-test"[tiab] OR "sister chromatid
exchange"[tiab] OR "SOS-response"[tiab] OR "polyploid*"[tiab] OR "genomic
instability"[tiab] OR "dna repair*"[tiab] OR "aneuploid*"[tiab]) NOT"Medline"[Filter]) OR "epigenesis, genetic"[mh] OR "epigenomics"[mh] OR"DNA methylation"[mh] OR "gene silencing"[mh] OR "histone
deacetylases"[mh] OR "RNA Interference"[mh] OR "microRNAs"[mh] OR "rna,small interfering"[mh] OR "cpg islands/genetics"[mh] OR "cpg island
methylator"[tiab] OR "cpg island methylation"[tiab] OR "epigenotype"[tiab] OR"epimutation*"[tiab] OR "methylation associated silencing"[tiab] OR "histone
tail modifications"[tiab] OR "histone tail modification"[tiab] OR "chromatinorganization"[tiab] OR "chromatin packag*"[tiab] OR "histone

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Search Terms

Notes

modification"[tiab] OR "histone retention"[tiab] OR "epigenetic*"[tiab] OR"epigenomic*"[tiab] OR "RNA Interference"[tiab] OR "noncoding rna"[tiab] OR"ncRNA"[tiab] OR "gene activation"[tiab] OR "proteasome"[tiab] OR "DNAmethylation"[tiab] OR "Methylation"[Title] OR "CIMP"[tiab] OR "FreeRadicals"[mh] OR "Reactive Oxygen Species"[mh] OR "Oxidative stress"[mh]OR "Electron Transport"[mh] OR "free radical*"[tiab] OR "oxygen
radical*"[tiab] OR "Oxidative stress"[tiab] OR "redox balance"[tiab] OR"oxidative damage*"[tiab] OR "Reactive Oxygen Species"[tiab] OR "reactive
nitrogen species"[tiab] OR "superoxide radical*"[tiab] OR "hydroxyl
radical"[tiab] OR "glutathione deplet*"[tiab] OR "oxidative protein
damage"[tiab] OR "C-reactive protein"[mh] OR "eosinophils"[mh] OR("fibrinogen"[tiab]AND "Inflammation"[tiab]) OR "chronicinflammation"[tiab] OR"chronically inflamed"[tiab] OR "acute inflammat*"[tiab] OR "infiltrating
leukocyt*"[tiab] OR "inflammatory-leukocyte"[tiab] OR "inflammatoryleukocytes"[tiab] OR "leukocyte infiltrat*"[tiab] OR "pro-inflammatory"[tiab] OR"proinflammatory"[tiab] OR "macrophage-recruitment"[tiab] OR "macrophageinflammatory proteins"[tiab] OR "macrophage colony stimulating factor*"[tiab]
OR "urethritis"[tiab] OR "prostatitis"[tiab] OR "seminal vesiculitis"[tiab] OR"epididymitis"[tiab] OR "orchitis"[tiab]
5("hypothalamic pituitary ovarian axis"[tiab] OR "hpo axis"[tiab] OR "gonadal
hormones"[mh] OR "pituitary hormones"[mh] OR "gonadotropin releasing
hormone"[mh] OR "follicle stimulating hormone"[mh] OR "testosterone"[mh]OR "receptors, gonadotropin"[mh] OR "receptors, pituitary hormone"[mh] OR"receptors, estrogen"[mh] OR "receptors, oxytocin"[mh] OR "estrogens"[mh]OR "estradiol"[mh] OR "estriol"[mh] OR "estrone"[mh] OR "luteinizing
hormone"[mh] OR "androgens"[mh] OR "hydroxyprogesterones"[mh] OR"Dehydroepiandrosterone"[mh] OR "Androstenedione"[mh] OR"Androstenediol"[mh] OR "Dihydrotestosterone"[mh] OR "androgen
receptor*"[tiab] OR "estradiol receptor*"[tiab] OR "estrogen receptor*"[tiab]
OR "follicle stimulating hormone"[tiab] OR "fsh receptor*"[tiab] OR"gonadotropin releasing hormone"[tiab] OR "hormone receptor*"[tiab] OR "lhreceptor*"[tiab] OR "luteinizing hormone"[tiab] OR "oestrogen receptor*"[tiab]
OR "ovarian hormone*"[tiab] OR "ovarian steroid*"[tiab] OR "oxytocin
receptor*"[tiab] OR "plasma membrane receptor*"[tiab] OR "prolactin
receptor*"[tiab] OR "reproductive hormone*"[tiab] OR "sex hormone*"[tiab] OR"testosterone receptor*"[tiab] OR "activin"[tiab] OR "estradiol"[tiab] OR"estriol"[tiab] OR "estrogen"[tiab] OR "estrone"[tiab] OR "FSH"[tiab] OR"gnrh"[tiab] OR "gonadotropin*"[tiab] OR "gonadotropin receptor*"[tiab] OR"hcg"[tiab] OR "inhibin"[tiab] OR "LH"[tiab] OR "LHRH"[tiab] OR "oestriol"[tiab]
OR "oestradiol"[tiab] OR "oestrogen"[tiab] OR "oestrone"[tiab] OR"Oxytocin"[tiab] OR "progesterone"[tiab] OR "prolactin"[tiab] OR"Steroidogenic"[tiab] OR "testosterone"[tiab] OR "Pregnenolone"[tiab] OR"17alpha hydroxy 6 methylene progesterone"[Supplementary Concept] OR"Dehydroepiandrosterone"[tiab] OR "DHEA"[tiab] OR "DHEAS"[tiab] OR"Androstenedione"[tiab] OR "Androstenediol"[tiab] OR

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Characteristics ofFemaleReproductive
Toxicity

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October 2021

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Search Terms

Notes"Dihydrotestosterone"[tiab] OR "steroidogenic acute regulatory
protein"[Supplementary Concept] OR "steroidogenic acute regulatory
protein"[tiab] OR "star protein"[tiab] OR "cholesterol side chain cleavage
enzyme"[mh] OR "cholesterol side chain cleavage enzyme"[tiab] OR"cholesterol desmolase"[tiab] OR "cytochrome p 450 scc"[tiab] OR"P450scc"[tiab] OR "CYP11A"[tiab] OR "CYP11A1"[tiab] OR "17alphahydroxylase"[tiab] OR "17,20 lyase"[tiab] OR "P450c17"[tiab] OR"CYP17"[tiab] OR "aromatase"[mh] OR "aromatase"[tiab] OR "cytochrome
p450 family 19"[mh] OR "cytochrome p450 family 19"[tiab] OR"P450arom"[tiab] OR "CYP19"[tiab] OR "3 or 17 beta hydroxysteroid
dehydrogenase"[Supplementary Concept] OR "3beta hydroxysteroid
dehydrogenase"[tiab] OR "3beta hsd"[tiab] OR "17beta hydroxysteroid
dehydrogenase"[tiab] OR "17beta hsd*"[tiab] OR "5alpha-reductase"[tiab] OR("DNA Adducts"[mh] OR "Comet Assay"[mh] OR "Germ-line mutation"[mh]OR "Mutagenesis"[mh] OR "Mutagenicity tests"[mh] OR "Sister-chromatid
exchange"[mh] OR "Mutation"[mh] OR "Ames-Assay"[tiab] OR "Amestest"[tiab] OR "Bacterial-Reverse-Mutation-Assay"[tiab] OR "clastogen*"[tiab]
OR "dna repair*"[tiab] OR "Genetic-toxicology"[tiab] OR "hyperploid"[tiab] OR"micronucleus-test"[tiab] OR "tetraploid"[tiab] OR "Chromosomeaberrations"[tiab] OR "DNA-damage"[tiab] OR "mutation*"[tiab] OR"chromosome-translocations"[tiab] OR "dna protein crosslink*"[tiab] OR "dnadamag*"[tiab] OR "dna inhibit*"[tiab] OR "Micronuclei"[tiab] OR"Micronucleus"[tiab] OR "Mutagens"[tiab] OR "strand break*"[tiab] OR"unscheduled dna synthes*"[tiab] OR "chromosomal aberration*"[tiab] OR"chromosome aberration*"[tiab] OR "chromosomal abnormalit*"[tiab] OR"chromosome abnormalit*"[tiab] OR "genotoxic*"[tiab] OR "adductformation"[tiab] OR "dna adduct*"[tiab] OR "dna break*"[tiab] OR "dsdna
break*"[tiab]) OR ("epigenesis, genetic"[mh] OR "epigenomics"[mh] OR "DNAmethylation"[mh] OR "gene silencing"[mh] OR "histone deacetylases"[mh] OR"RNA Interference"[mh] OR "microRNAs"[mh] OR "rna, small interfering"[mh]OR "cpg islands/genetics"[mh] OR "cpg island methylator"[tiab] OR "cpgisland methylation"[tiab] OR "epigenotype"[tiab] OR "epimutation*"[tiab] OR"methylation associated silencing"[tiab] OR "histone tail modifications"[tiab]
OR "histone tail modification"[tiab] OR "chromatin organization"[tiab] OR"chromatin packag*"[tiab] OR "histone modification"[tiab] OR "histone
retention"[tiab] OR "epigenetic*"[tiab] OR "epigenomic*"[tiab] OR "RNAInterference"[tiab] OR "noncoding rna"[tiab] OR "ncRNA"[tiab] OR "geneactivation"[tiab] OR "proteasome"[tiab] OR "DNA methylation"[tiab] OR"Methylation"[Title] OR "CIMP"[tiab]) OR ("mitochondria"[mh] OR "oxidative
phosphorylation"[mh] OR "mitochrondria*"[tiab] OR "oxidative
phosphorylation"[tiab] OR "oxidative damage"[tiab] OR "fatty acid betaoxidation"[tiab] OR "calcium buffering"[tiab] OR "ca2 buffering"[tiab] OR"mitochondrial dna mutation*"[tiab] OR "mtdna mutation*"[tiab] OR"mtDNA"[tiab]) OR ("Free Radicals"[mh] OR "Reactive Oxygen Species"[mh]OR "Oxidative stress"[mh] OR "Electron Transport"[mh] OR "freeradical*"[tiab] OR "oxygen radical*"[tiab] OR "Oxidative stress"[tiab] OR

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Search Terms

Notes"redox balance"[tiab] OR "oxidative damage*"[tiab] OR "Reactive Oxygen
Species"[tiab] OR "reactive nitrogen species"[tiab] OR "superoxide
radical*"[tiab] OR "hydroxyl radical"[tiab] OR "glutathione deplet*"[tiab] OR"oxidative protein damage"[tiab]) OR ("cytotoxicity, immunologic"[mh] OR"Immunologic Factors"[mh] OR "Immunomodulation"[mh] OR "B-Cell
Activation Factor Receptor"[mh] OR "Antigenic Modulation"[mh] OR "B-Cell
Activating Factor"[mh] OR "Immunologic Factors"[Pharmacological Action]
OR "b-cell-activation"[tiab] OR "immune surveillance"[tiab] OR "immune
suppress*"[tiab] OR "immunostimulant"[tiab] OR "immune-activation"[tiab] OR"immunodeficien*"[tiab] OR "somatic-hypermutation"[tiab] OR "immuneactivation"[tiab] OR "immune-system-activation"[tiab] OR "Chronic-antigenicstimulation"[tiab] OR "immunosuppress*"[tiab] OR "immune
dysregulation"[tiab]) OR ("signal transduction"[mh] OR "signaltransduction"[tiab] OR "signal pathway*"[tiab] OR "signaling pathway*"[tiab]
OR "ion channel"[tiab] OR "signaling system*"[tiab] OR "cell signal*"[tiab] OR"cellular signal*"[tiab] OR "intracellular signal*"[tiab] OR "signal
cascade*"[tiab] OR "signaling cascade*"[tiab] OR "second messenger*"[tiab]
OR "calcium signal*"[tiab]) OR ("cell communication"[mh] OR "gapjunctions"[mh] OR "connexins"[mh] OR "connexins"[Supplementary Concept]
OR "cell communication*"[tiab] OR "cellular communication*"[tiab] OR"intracellular communication*"[tiab] OR "cell interaction*"[tiab] OR "gapjunction*"[tiab] OR "connexin*"[tiab]) OR ("Apoptosis"[mh] OR "cytotoxicity,
immunologic"[mh] OR "Caspases"[mh] OR "autophagy"[mh] OR"necrosis"[mh] OR "Autolysis"[mh] OR "Angiogenesis Modulating Agents"[mh]OR "Angiogenesis Inducing Agents"[Pharmacological Action] OR"Angiogenesis Inducing Agents"[mh] OR "neovascularization, pathologic"[mh]OR "Cell Proliferation"[mh] OR "homeostasis"[mh] OR "Cyclin-Dependent
Kinases"[mh] OR "Cyclin-Dependent Kinase Inhibitor Proteins"[mh] OR"Mitogens"[mh] OR "Mitogens"[Pharmacological Action] OR "cell hypoxia"[mh]OR "angiogenic"[tiab] OR "Apoptosis"[tiab] OR "autophagy"[tiab] OR"Caspases"[tiab] OR "cell cycle control*"[tiab] OR "cell cycle arrest"[tiab] OR"cell hypoxia"[tiab] OR "Cell Proliferation"[tiab] OR "cellular-energetics"[tiab]
OR "cellular-hypoxia"[tiab] OR "cellular proliferation"[tiab] OR "cellular
replication*"[tiab] OR "Cytogenesis"[tiab] OR "Cytogenic"[tiab] OR"Cytotoxin"[tiab] OR "hepatocellular-proliferation"[tiab] OR "hyperplasia"[tiab]
OR "hypoxic cell*"[tiab] OR "mitogenesis"[tiab] OR "mitotic checkpoint*"[tiab]
OR "Neoplasia"[tiab] OR "p53 delet*"[tiab] OR "p53 inactivat*"[tiab] OR "p53inhibit*"[tiab] OR "prb delet*"[tiab] OR "prb inactivat*"[tiab] OR "prbinhibit*"[tiab] OR "programmed cell death"[tiab] OR ("Rb"[All Fields] AND"p16ink4a inactiv*"[tiab]) OR "retinoblastoma-protein"[tiab] OR"senescence"[tiab] OR "senescent"[tiab] OR "survivin"[tiab]) OR"microtubules"[mh] OR "spindle apparatus"[mh] OR "microtubule organizing
center"[mh] OR "microtubule*"[tiab] OR "spindle formation"[tiab] OR "spindle
apparatus"[tiab] OR "meiotic spindle*"[tiab] OR "mitotic spindle*"[tiab])

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Search Terms

Notes

6

#2 OR #3 OR #4 OR #5

Combine DART
concept groups

7#1 AND #6

CombineChemical &DART

8("Animals, Genetically Modified"[mh] OR "Animals, Inbred Strains"[mh] OR"Chimera"[mh] OR "Animals, Laboratory"[mh] OR animals[mh:noexp]) OR(animal-stud*[tiab] OR wood-mouse[tiab] OR murinae[tiab] OR muridae[tiab]
OR cricetinae[tiab] OR rodentia[tiab] OR rodent[tiab] OR rodents[tiab] ORferrets[tiab] OR ferret[tiab] OR polecat*[tiab] OR mustela-putorius[tiab] ORcavia[tiab] OR callithrix[tiab] OR marmoset*[tiab] OR chinchilla*[tiab] ORjird[tiab] OR jirds[tiab] OR merione[tiab] OR meriones[tiab] OR cats[tiab] ORcat[tiab] OR felis[tiab] OR canis[tiab] OR sheep[tiab] OR sheeps[tiab] ORgoats[tiab] OR goat[tiab] OR capra[tiab] OR saguinus[tiab] OR tamarin*[tiab]
OR leontopithecus[tiab] OR ape[tiab] OR apes[tiab] OR pan-paniscus[tiab]
OR bonobo*[tiab] OR pan-troglodytes[tiab] OR gibbon*[tiab] ORsiamang*[tiab] OR nomascus[tiab] OR symphalangus[tiab] ORchimpanzee*[tiab] OR orangutan*[tiab] OR horse[tiab] OR horses[tiab] ORequus[tiab] OR cow[tiab] OR cows[tiab] OR chicken[tiab] OR chickens[tiab]
OR wistar[tiab] OR balb[tiab] OR C57[tiab] OR C57bl[tiab] OR quail[tiab] ORlong-evans[tiab] OR guppy[tiab] OR medaka[tiab] OR zebrafish[tiab] ORflying-fox[tiab] OR Fruit-bat[tiab] OR non-human-primate*[tiab] OR
capuchin*[tiab] OR rhesus[tiab] OR macaque*[tiab] OR cattle[tiab] ORbovine[tiab] OR pigs[tiab] OR pig[tiab] OR swine[tiab] OR swines[tiab] ORpiglet*[tiab] OR Sprague-Dawley[tiab] OR vervet*[tiab]) OR ((mice[tiab] ORmouse[tiab] OR murine[tiab] OR rats[tiab] OR rat[tiab] OR hamster[tiab] OR
hamsters[tiab] OR guinea-pig*[tiab] OR gerbil*[tiab] OR rabbits[tiab] OR
rabbit[tiab] OR dogs[tiab] OR dog[tiab] OR monkey[tiab] OR monkeys[tiab]
OR pongo-pygmaeus[tiab] OR sow[tiab] OR sows[tiab] OR boar[tiab] ORboars[tiab]) NOT medline[sb]) OR ((in vitro[tiab] OR in vitro techniques[mh]OR cell line*[tiab]) AND animals[mh:noexp])

9

Experimental
animal terms(Modified from
RoC)

CombineChemical +DART + Animals#7 AND #8

FINAL

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