25/10/2022
Proposal for reporting data relevant for human health
Human Health Assessment of Perfluoroalkyl carboxylic Acids (PFCAs)
Current state of the assessment based on in vivo studies used in the EFSA Opinion (2020)
and ATSDR Report (2018). The assessment is ongoing with more recent studies and
epidemiological studies.

Commented [A1]: Do we have a strict format to
follow – restriction dossier format – how much
can we deviate from that format?
When we have agreed on a format I believe we
should combine all the PFASs in one doc?
Field Code Changed
Field Code Changed

Content:
1.

Toxicokinetics ____________________________________________________________ 2

2.

Repeated dose toxicity _____________________________________________________ 3

3.

Developmental and reproductive toxicity _____________________________________ 5

4.

Carcinogenicity ___________________________________________________________ 7

5.

References _______________________________________________________________ 9

6.

Annex 1: Toxicokinetic studies______________________________________________ 18

7.

Annex 2: Repeated dose toxicity studies _____________________________________ 19

8.

Annex 3: Developmental and reproductive toxicity studies ______________________ 73

9.

Annex 4: Carcinogenicity studies____________________________________________ 93

1

1. Toxicokinetics
Work in progress and to be added later.

Commented [A2]: What is the planned to be
included here? We suggest summarizing
experimental animal and human information
separately based on the EFSA opinion without
tabling individual studies and only add new
studies not covered

2

2. Repeated dose toxicity
Epidemiological studies

Commented [A3]: To put after experimental
animal tox, organized by endpoint.

To be completed…
Animal studies (summarised in Table 2)
Oral exposure to PFOA by gavage induces strong adverse effects in rodent liver morphology, immune
function, thyroid morphology/metabolism, and clinical chemistry, especially reduced cholesterol and
triglycerides.

Commented [A4]: Should we rather organize by
endpoint and not substance?

Strong increases in liver weight are observed after exposure to 0.625 mg/kg bw/d or 1 mg/kg bw/d
and higher in male rats and mice, respectively (NTP, 2019b). In female rats liver weights increased at
25 mg/kg bw/d and higher.
Immunotoxic effects of PFOA include changes in weights of lymphoid organs (thymus, spleen),
cellularity of white blood cells, and antibody responses to T-cell-dependent antigens. From
immunological studies performed in male mice and rats, the male rat seems to be less sensitive
(Loveless et al., 2008). In rats, thymic or splenic weight was reduced at 10 or 2.5 mg/kg bw/d or
higher in males (NTP, 2019b); female rats have not been studied so far. Thymic weight was reduced
in male or female mice at doses of 7.5 mg/kg bw/d or higher (DeWitt et al., 2016; Qazi et al., 2012);
splenic weight was reduced at doses of 1 or 3.75 mg/kg bw/d or higher in male or female mice,
respectively (DeWitt et al., 2008; Loveless et al., 2008). Antibody responses were reduced at doses of
10 or 1.88 mg/kg bw/d or higher in male or female mice, respectively (DeWitt et al., 2016; Loveless
et al., 2008); no effects on antibody response were observed in male rats (Loveless et al., 2008).
Numbers of white blood cells were altered doses of 0.49 and 3.75 mg/kg bw/d or higher in male and
female mice, respectively (DeWitt et al., 2016; Son et al., 2008; Son et al., 2009).
PFOA induced an increase in thyroid weight at 1.25 mg/kg bw/d or higher and a reduction of serum
thyroid hormones T3 and T4 at 0.625 or 100 mg/kg bw/d or higher in male and female rats (NTP,
2019b). Likewise, thyroid hormones were reduced in male in Cynomolgus monkeys at a dose of 20-30
mg/kg bw/d for T3, and at 3 or 10 mg/kg bw/d or higher for total or free T4 (Butenhoff et al., 2002).
With respect to clinical chemistry, reductions in cholesterol and triglycerides in blood serum are the
most prominent findings for PFOA. Cholesterol was reduced at a LOAEL of 0.3 mg/kg bw/d in male
rats (Loveless et al., 2006); female rats have not been studied. In male mice, PFOA reduced
cholesterol at a LOAEL of 5 mg/kg bw/d (Wu et al., 2018); in female mice, 15 mg/kg bw/d is the
lowest dose at which PFOA reduced cholesterol (DeWitt et al., 2009). Serum triglycerides were
reduced at a LOAEL of 0.3 mg/kg bw/d in male rats and mice (Loveless et al., 2008); no data are
available for female rats. Female mice showed reduced triglycerides at a LOAEL of 3.75 mg/kg bw/d
(DeWitt et al., 2009).
PFNA, PFDA, PFUnDA and PFDoDA raise similar concerns in many of the effects described above in
male and female rats and mice compared to repeated exposure to PFOA. Potencies are in
comparable dose ranges as PFOA for the respective effect (Ding et al., 2009)Fang et al., 2008;
Frawley et al., 2018; Harris and Birnbaum 1989; Kato et al., 2015; Kudo et al., 2006; NTP, 2019; Shi et
al., 2009; Takahashi et al., 2014; Wang et al., 2015; Zhang et al., 2008).
Short chain PFCAs (C2: TFA, C4: PFBA, C6: PFHxA, C7: PFHpA) as well as long-chain (C14-C18) PFCAs
(C14: PFTeDA, C16: PFHxDA, C18: PFODA) all induced increased liver weight, but LOAELs are (much)
higher (10-1000-fold) when compared to C8-C12. Generally, at very short (TFA) or very long (PFODA)
chain length, LOAELs are highest and only few effects have been reported (reduced body weight,
3

Commented [A5]: Immunotoxicity should be a
separate chapter perhaps- or should it be a
subchapter under repeated dose? in order to
avoid to much repetition its possible to refer to
the different chapters and just describe it briefly
here. Same goes for the endocrine part- perhaps
we should have a separate chapter for endocrine
disruptive effects as well.
Commented [A6]: Why this endpoint emphasized
for PFOA? More relevant for PFOS (and for PFAS
as group)
Commented [A7]: Loveless 2008 included only
males, so cannot address sex-differences. They
showed that male rats were much less sensitive
than male mice. We suggest to move “male”
before mice and delete male here

increased liver weight and for PFODA reduced cholesterol are the only significant effects). The most
sensitive effects for short-chain (PFBA, PFHxA, PFHpA) and long-chain (PFTeDA, PFHxDA) PFCAs are
those on the thyroid system (PFBA: Butenhoff et al., 2012a; PFHxA: NTP, 2019a; PFHpA: Anonymous,
2017; PFTeDA & PFHxDA: Hirata-Koizumi et al., 2015).
Research gaps
No data are available for PFPA (C3), PFPeA (C5), PFTrDA (C13), PFPeDA (C15) and PFHpDA (C17).
Only one study is available for C2, C11, C14, C16, C18 each, and only for rats. Immunotoxicology in
general is mainly studied in terms of lymphatic tissues (spleen and thymus) morphology and white
blood cell count, but immunological effects of PFCAs can be much more complex as demonstrated in
mice studies on PFOA (DeWitt et al., 2016; Loveless et al., 2008; Son et al., 2008, Son et al., 2009).
Especially antibody responses have only been studied in mice after exposure to PFOA.
Dermal and inhalation application routes have been studied to a much more limited extent
compared to the oral gavage route. Thus, there is a major research gap with respect to inhalation
and dermal administration. The few available studies, however suggest that for these routes, affects
are similar to those observed for the oral route (reduced body weight, increased liver weight,
decreased weight of lymphatic tissues) after PFOA administration via these exposure routes (dermal:
Kennedy et al., 1985; Fairley et al., 2007; O’Malley and Ebbins, 1981; inhalation: Kennedy et al.,
1986).
In conclusion, there are overlaps in the type of adverse effects across all PFCAs but there are also
marked differences in the potencies of PFCA to induce adverse effects on rodent physiology. Besides
gradual variations in the magnitudes of identical effects, TFAA and PFODA lack some major health
hazards that apply for PFOA and other PFCA.

4

Commented [A8]: Gaps only for repeat tox?
Should come in the end or for each endpoint?

Commented [A9]: Even for PFOA the investigation
is quite poor. Should be studied in a comparative
way across PFCAs and PFSAs. To describe in a
chapter on research gaps later

Commented [A10]: Need to say something on
potency differences is dependent on study
duration and tissue concentration (internal
versus external dose), but perhaps that is
planned in the TK part? And perhaps it belongs
more in an overall consideration across PFCAs
and PFSAs

3. Developmental and reproductive toxicity
Epidemiology
Legacy PFAS, including, PFOA and PFOS have been associated with adverse effects on reproduction
and development in epidemiological studies. Observed effects comprised decreased sperm counts,
higher levels of luteinizing and follicle stimulation hormone (Vested et al., 2013; Song et al., 2018)
and deteriorated semen parameters such as mobility and DNA fragmentation ratio (Pan et al., 2019).
Exposure to PFOA was linked to reduced fecundity (Velez et al., 2015) or reduced odds for
fecundability (Fei et al., 2009). Further, there is evidence for decreased infant weight in relation to
PFOA exposure (Johnson et al., 2014), as well as delayed menarche (Kristensen et al., 2013).
Although reported human and epidemiological data is not always consistent (Bach et al., 2016),
systematic reviews of available studies indicated adverse effects of environmental exposure to legacy
PFAS on both female and male reproduction (Fenton et al., 2019). This is further supported by
assessments of human health hazards, based on in vivo studies in rodents of developmental and
reproductive toxicity as listed in the attached table (Table 3).

Commented [A11]: Should rather be based on
EFSA and expanded with new studies. Put
epidemiology after animal tox, cannot be divided
by substance type

Animal studies (summarised in Table 3)
Mice are regarded as the most sensitive species, showing severe developmental and reproductive
effects after exposure to PFOA in utero. In particular, neonatal morbidity and reduced birth weights
are observed after exposure to 5 mg/kg bw/d (Lau et al., 2006; Song et al., 2018; White et al., 2011;
Yahia et al., 2010) or lower (starting at 0.6 mg/kg bw/d), depending on strain (Abbot et al., 2007;
Tucker et al., 2015). Further, a substantial increase of full litter resorptions (FLR) was observed at a 5
mg/kg bw exposure levels. Concomitantly, placental lesions, impaired morphology and deteriorated
functional scores of the mammary glands were also observed (Macon et al., 2011; Tucker et al.,
2015).
With respect to foetal and/or neonatal mortality, developmental toxicities of PFNA and PFDA were
similar to that of PFOA, because these effects were observed at comparable exposure levels. For
PFNA, neonatal survival was reduced down to 20% on PND 10 at 5 mg/kg bw/d (Das et al., 2015).
Further, serum testosterone and other parameters of male fertility were affected by PFNA at a LOAEL
as low as 0.5 mg/kg bw/d in mice (Singh and Singh, 2019a). PFDA exposure decreased the ratio of
viable foetuses and increased litter resorption at a LOAEL level of 12.8 mg/kg bw/d in mice (Harris
and Birnbaum, 1989). The LOAEL for reduced foetal weight was comparatively low (PFDA: 0.5 mg/kg
bw/d GD10-13).
One study on PFUnDA demonstrated reduced birth weights in male (13.4%) and female (12.5%) rat
pups, at an exposure level of 1 mg/kg bw/d (Takahashi et al., 2014). However, no other significant
effects on reproduction or development were reported.
For PFDoDA parental body weight was reduced at LOAEL levels ranging between 0.5 – 3 mg/kg bw/d
in rats (Chen et al., 2019; Shi et al., 2009a; Shi et al., 2009b). Further, mortality of pregnant female
rats was increased at an LOAEL of only 2.5 mg/kg bw/d (Kato et al., 2015). PFDoDA did also affect
expression of ovarian or testicular genes in rats (LOAEL of 0.5-3 mg/kg bw/d for females or 0.02-0.5
mg/kg bw/d for males), which led to decreased levels of reproductive hormones (LOAEL 5 mg/kg
bw/d or below) and reduced weight of testes (LOAEL 10 mg/kg bw/d). This confirms substantial toxic
effects on development and reproduction. However, comparison with PFOA is difficult as data on
mice are lacking.
Exposure of up to 100 mg PFTeDA per kg bw/d did not affect oestrous cyclicity, fertility indices or
other parameters of reproduction in rats (Hirata-Koizumi et al., 2015). Minor, but significantly
5

Commented [A12]: This is occurring at much
lower levels than other PFOA effects, needs to be
described. Only studied for PFOA. Applies both to
neonatal development and delay in involution in
the dam at weaning (developmental effect in
both immature and adult animals).

reduced maternal body weights have been observed during (LOAEL 3 mg/kg bw/d) and after
lactation (10 mg/kg bw/d), as well as in mated males and pups (10 mg/kg bw/d).
Treatment of rats with PFHxDA resulted in reduced paternal body weights at a much higher LOAEL of
100 mg/kg bw/d. No other effects in relation to reproduction or development were observed. PFODA
did affect both parental and pup body weights and fertility parameters, such as number of
implantations or number of pups. The LOAEL for adversities on reproduction and development was
1000 mg/kg.
Compared to PFOA, PFHxA and PFBA are less potent in respect to effects on reproduction and
development. For example, the NOAEL for pup mortality related to PFHxA exposure during
pregnancy was 175 mg/kg bw/d (Iwai et al., 2019). Reduced birth weight or neonatal weight were
observed at a LOAEL of 500 mg/kg bw/d. For PFBA, no increased foetal or pup mortality, reduced
implantation or increased FLR was observed. Some mild developmental effects, such as slightly
delayed eye opening (LOAEL 35 mg/kg bw/d), were reported.
In conclusion, there are overlaps in the type of adverse effects across all PFCAs but marked
differences in the potencies of PFCA to induce adverse effects on development and reproduction.
Besides gradual variations in the magnitudes of identical effects, PFBA, PFHxDA and PFODA lack some
major health hazards that apply for PFOA and other C6-C14 chained PFCA. For both PFHxDA and
PFODA, this could be related to an insufficient availability in potential target tissues or organs.

6

Commented [A13]: Add species

Commented [A14]: This needs to be further
described when toxicokinetic analysis is finished.

Commented [A15]: Due to the limited data, we
described in the text for carcinogenicity not only
PFCAs but also other PFAS. For this endpoint it
probably does not make much sense to discuss it
per group?

4. Carcinogenicity
Current classifications
Several PFAS have previously been classified as possibly or probably carcinogenic to humans. The
International Agency for Research on Cancer (IARC) classified PFOA as possibly carcinogenic (Group
2B; (IARC, 2017)) and tetrafluoroethylene as probably carcinogenic to humans (Group 2A; (IARC,
2017)), while other PFAS, such as 2-Chloro-1,1,1-trifluoroethane, are considered as not classifiable
due to lack of data. The U.S. Environmental Protection Agency (EPA) found that there is suggestive
evidence that PFOA (EPA US, 2016b), PFOS (EPA US, 2016a), and GenX (EPA US, 2018) may cause
cancer. The EFSA Contam Panel concluded in 2018 that there is insufficient support for
carcinogenicity of PFOS and PFOA in humans from epidemiological studies but that both compounds
induced tumours in rats (EFSA, 2020b). According to the REACH regulation in the EU, 17 PFAS are
currently harmonised classified as carcinogenic (Carc. 2 or Carc. 1B; e.g., PFOA and its ammonium
salt, PFNA and its sodium and ammonium salts, PFDA and its sodium and ammonium salts, PFOS and
it ammonium, lithium and potassium salts, trifluralin). Additionally, amongst 6790 PFAS registered in
the EU, 76 PFAS are self-classified by registrants as Carc. 1A/B or Carc. 2.
Animal studies (summarised in Table 4)
Chronic repeated-dose toxicity studies performed in mammalian animals have been performed with
PFHxA (Klaunig et al., 2015), PFOA (3M, 1983; Biegel et al., 2001; NTP, 2019a), PFOS (Butenhoff et al.
2012), GenX chemicals (EPA US, 2018; Temkin et al., 2020), and tetrafluoroethylene (IARC, 2017).
For PFHxA, there was no evidence for tumorigenic activity in SD rats (Klaunig et al., 2015).
For PFOA, 24 months repeated-dose toxicity studies with rats resulted in an increased incidence of
Leydig cell adenoma (3M, 1983; Biegel et al., 2001), fibroadenoma in mammary glands (3M, 1983),
liver adenoma (Biegel et al., 2001), and acinar cell adenoma (Biegel et al., 2001). A re-evaluation of
the original slides of the 3M study by a Pathology Working Group resulted in no significant increase
of fibroadenomas (Hardisty et al., 2010). A re-evaluation of male tissue samples of the 3M study by
Caverly-Rae et al. (2014) resulted in an increase in acinar cell hyperplasia but not adenoma or
carcinoma. In a more recent 2-year diet carcinogenicity study by the US-NTP (2019) with SD rats
starting with prenatal exposure, the induction of pancreatic tumours (acinar cell adenomas and
adenocarcinomas) as well as hepatocellular adenoma and carcinoma was confirmed at dose levels as
low as 1.1 mg/kg bw/d. Additionally, PFOA was identified as tumour promoting in rat liver (Abdellatif
et al., 1991) and trout liver (Benninghoff et al., 2012). Besides PFOA, also PFNA and PFDA showed
tumour promoting activity in the trout study (Table 4).
Epidemiology
A variety of epidemiological studies, i.e., worker, high exposure community, and general population
studies, assessed possible associations between PFAS exposure and cancer risk. These studies have
been summarised in previous reports (ATSDR, 2018; EFSA, 2020a; EFSA, 2020b; IARC, 2017). In some
studies PFOA exposure was associated with increased risks of testicular (Barry et al., 2013), kidney
(Steenland and Woskie, 2012; Vieira et al., 2013) and prostate cancer (Gilliland and Mandel, 1993;
Hardell et al., 2014; Lundin et al., 2009). In the study by Hardell et al. (2014), a statistically significant
association between PFAS exposure and prostate cancer was also observed for PFDeA, PFUA , PFHxS,
and PFOS. Inverse associations (i.e., decreased risk) were observed between PFOA exposure and
bladder (Steenland et al., 2015), colorectal (Innes et al., 2014) and breast cancer (Barry et al., 2013).
According to the EFSA CONTAM Panel (2018) these studies provide insufficient support for
7

Commented [A16]: Should we have this
description for the other chapters as well?

carcinogenicity of PFOS and PFOA in humans. The IARC assessment of PFOA (2017) concluded that
the epidemiological evidence for increased testicular cancer risk through PFOA exposure is credible
and unlikely to be explained by bias and confounding factors, however, it is based on a small number
of testicular cancer cases (n=17; Barry et al., 2013). Also the increased kidney cancer risk through
PFOA exposure is considered credible by IARC (IARC, 2017). IARCS overall conclusion was that there is
limited evidence in humans for the carcinogenicity of PFOA despite the positive associations for
cancers of the testis and kidney. It should be emphasized that the associations between testis cancer
and PFOA exposure are in line with increased incidence of Leydig cell adenoma in animal studies (3M,
1983; Biegel et al., 2001).
Mode of Action
In a recent evaluation of the likely mechanisms of tumour formation by PFAS, Temkin and coworkes
identified, that multiple PFAS induce oxidative stress, are immunosuppressive, and modulate
receptor-mediated effects (Temkin et al., 2020). They also found suggestive evidence that some PFAS
can induce epigenetic alterations and influence cell proliferation, while genotoxic mechanisms and
metabolic activation is not supported by current scientific evidence.
For PFOA, the PPARα-agonistic mode of action is a plausible explanation for liver carcinogenicity as
observed in rats (ATSDR, 2018). However, also interactions with hepatic estrogen receptors are
discussed as potential PPARα-independent mechanisms for liver carcinogenicity (ATSDR, 2018).
According to ATSDR, “the induction of Leydig cell tumors by PFOA may be mediated by effects on
aromatase activity or testosterone biosynthesis, both of which may be related to PPARα activation”.
In the Background document to the Opinion on the Annex XV dossier proposing restrictions on PFOA,
PFOA salts and PFOA-related substances, the Committee for Risk Assessment concluded that despite
human PPARα does not seem to be involved in the induction of cell proliferation in the liver
(Klaunig et al., 2012), PFOA-induced rat liver tumours cannot be regarded as irrelevant for
humans (RAC 2015).
According to the EFSA Opinion on PFAS, the Leydig cell tumour induction by PFOA could be a result of
“reduced serum testosterone levels and compensatory releases of luteotrophic hormone, which
stimulates growth of Leydig cells and tumour formation.” Also the induction of pancreatic acinar
tumours by PFOA may be related to PPARα-agonistic activity. The Committee for Risk Assessment
concluded that the data are insufficient to characterize the mode of action of PFOA induced Leydig
cell adenomas and pancreatic acinar cell tumours and hence carcinogenic effects at these sites are
presumed to be relevant for humans (RAC 2015).
In conclusion, for tetrafluoroethylene, PFOA, PFOS and GenX there is evidence for carcinogenic
effects from animal studies but no clear evidence for carcinogenicity in humans. For the observed
carcinogenicity, the available information is not sufficient to rule out human relevance of the
underlying mode of action. According to the REACH regulation in the EU, 17 PFAS are currently
harmonised classified as carcinogenic, partly based on read across to PFOA. For the vast majority of
PFAS, long-term toxicity or carcinogenicity studies as well as epidemiological studies informative on
potential carcinogenic effects are not available and thus, carcinogenicity of most PFAS is unclear.

8

Commented [A17]: I see this MOA as linked only
to carcinogenicity? We could consider to have a
MOA discussion across PFASs in the end instead
of separating by the classes.
Of note, in the EFSA opinion the MoA was
restricted to the most sensitive endpoints, so not
everything is covered. Perhaps we can also here
focus on the most prominent effects.
PPARa activation is relevant for human
physiology and should not be discussed only in
light of liver tumours.
Commented [A18]: Do we need a separate MoA
search or is it likely to be covered by the main
search for each PFAS-group?

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9

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Singh S. and Singh S.K. (2019b): Chronic exposure to perfluorononanoic acid impairs spermatogenesis,
steroidogenesis and fertility in male mice. J Appl Toxicol 39 (3), 420-431. DOI: 10.1002/jat.3733
Singh S. and Singh S.K. (2019c): Effect of gestational exposure to perfluorononanoic acid on neonatal mice
testes. J Appl Toxicol 39 (12), 1663-1671. DOI: 10.1002/jat.3883
Singh S. and Singh S.K. (2019d): Prepubertal exposure to perfluorononanoic acid interferes with
spermatogenesis and steroidogenesis in male mice. Ecotoxicol Environ Saf 170, 590-599. DOI:
10.1016/j.ecoenv.2018.12.034

15

Son H.Y., Kim S.H., Shin H.I., Bae H.I., and Yang J.H. (2008): Perfluorooctanoic acid-induced hepatic toxicity
following 21-day oral exposure in mice. Archives of Toxicology 82 (4), 239-246. DOI: 10.1007/s00204-007-0246x
Son H.Y., Lee S., Tak E.N., Cho H.S., Shin H.I., Kim S.H., and Yang J.H. (2009): Perfluorooctanoic acid alters T
lymphocyte phenotypes and cytokine expression in mice. Environ Toxicol 24 (6), 580-588. DOI:
10.1002/tox.20459
Song P., Li D., Wang X., and Zhong X. (2018): Effects of perfluorooctanoic acid exposure during pregnancy on
the reproduction and development of male offspring mice. Andrologia 50 (8), e13059. DOI: 10.1111/and.13059
Steenland K. and Woskie S. (2012): Cohort mortality study of workers exposed to perfluorooctanoic acid. Am J
Epidemiol 176 (10), 909-917. DOI: 10.1093/aje/kws171
Steenland K., Zhao L., and Winquist A. (2015): A cohort incidence study of workers exposed to
perfluorooctanoic acid (PFOA). Occup Environ Med 72 (5), 373-380. DOI: 10.1136/oemed-2014-102364
Takahashi M., Ishida S., Hirata-Koizumi M., Ono A., and Hirose A. (2014): Repeated dose and
reproductive/developmental toxicity of perfluoroundecanoic acid in rats. J Toxicol Sci 39 (1), 97-108. DOI:
10.2131/jts.39.97
Tan X., Xie G., Sun X., Li Q., Zhong W., Qiao P., Sun X., Jia W., and Zhou Z. (2013): High fat diet feeding
exaggerates perfluorooctanoic acid-induced liver injury in mice via modulating multiple metabolic pathways.
PLoS One 8 (4), e61409. DOI: 10.1371/journal.pone.0061409
Temkin A.M., Hocevar B.A., Andrews D.Q., Naidenko O.V., and Kamendulis L.M. (2020): Application of the key
characteristics of carcinogens to per and polyfluoroalkyl substances. International Journal of Environmental
Research and Public Health 17 (5). DOI: 10.3390/ijerph17051668
Thomford P.J. (2001): 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in
Cynomolgus Monkeys. Covance 6329-231 / 3M T-6889.3. Covance Laboratories Inc.
Tucker D.K., Macon M.B., Strynar M.J., Dagnino S., Andersen E., and Fenton S.E. (2015): The mammary gland is
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van Otterdijk F.M. (2007a): Project 470677: Repeated dose 28-day oral toxicity study with MTDID-8391 by daily
gavage in the rat, followed by a 21-day recovery period. 3M, Maplewood, MN
van Otterdijk F.M. (2007b): Project 484492: Repeated dose 90-day oral toxicity study with MTDID 8391 by daily
gavage in the rat followed by a 3-week recovery period. 3M, Maplewood, MN
Vélez M.P., Arbuckle T.E., Fraser W.D. (2015): Maternal exposure to perfluorinated chemicals and reduced
fecundity: the MIREC study. Hum Reprod. 30:701-9. DOI: 10.1093/humrep/deu350.
Vested A., Ramlau-Hansen C.H., Olsen S.F., Bonde J.P., Kristensen S.L., Halldorsson T.I., Becher G., Haug L.S.,
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quality and reproductive hormones in adult men. Environ Health Perspect. 453-8. DOI: 10.1289/ehp.1205118.
Vetvicka V. and Vetvickova J. (2013): Reversal of perfluorooctanesulfonate-induced immunotoxicity by a
glucan-resveratrol-vitamin C combination. Oriental Pharmacy and Experimental Medicine 13 (1), 77-84. DOI:
10.1007/s13596-013-0105-7
Vieira V.M., Hoffman K., Shin H.M., Weinberg J.M., Webster T.F., and Fletcher T. (2013): Perfluorooctanoic acid
exposure and cancer outcomes in a contaminated community: a geographic analysis. Environ Health Perspect
121 (3), 318-323. DOI: 10.1289/ehp.1205829
Wang J., Yan S., Zhang W., Zhang H., and Dai J. (2015): Integrated proteomic and miRNA transcriptional analysis
reveals the hepatotoxicity mechanism of PFNA exposure in mice. J Proteome Res 14 (1), 330-341. DOI:
10.1021/pr500641b
Wang L., Zhao F., Kan M., Wen Z., Zhou Y., Yu L., and Liu H. (2017): [Effects of perfluorooctanoic acid on
oxidative stress and PPARalpha and its related CYP4A1 gene expression in rat liver]. Wei Sheng Yan Jiu 46 (5),
802-806. https://www.ncbi.nlm.nih.gov/pubmed/29903312

16

White S.S., Stanko J.P., Kato K., Calafat A.M., Hines E.P., and Fenton S.E. (2011): Gestational and chronic lowdose PFOA exposures and mammary gland growth and differentiation in three generations of CD-1 mice.
Environ Health Perspect 119 (8), 1070-1076. DOI: 10.1289/ehp.1002741
WIL R.L. (2005): A combined 28-day repeated dose oral toxicity study with the reproduction/developmental
toxicity screening test of perfluorohexanoic acid and 1H, 1H, 2H, 2H tridecafluoro-1-octanol in rats, with
recovery. Study number WIL-534001
Wolf C.J., Zehr R.D., Schmid J.E., Lau C., and Abbott B.D. (2010): Developmental effects of perfluorononanoic
Acid in the mouse are dependent on peroxisome proliferator-activated receptor-alpha. PPAR research 2010,
282896. DOI: 10.1155/2010/282896
Wolf D.C., Moore T., Abbott B.D., Rosen M.B., Das K.P., Zehr R.D., Lindstrom A.B., Strynar M.J., and Lau C.
(2008): Comparative Hepatic Effects of Perfluorooctanoic Acid and WY 14,643 in PPAR-α Knockout and Wildtype Mice. Toxicologic Pathology 36 (4), 632-639. DOI: 10.1177/0192623308318216
Wu X.M., Xie G.J., Xu X.X., Wu W., and Yang B. (2018): Adverse bioeffect of perfluorooctanoic acid on liver
metabolic function in mice. Environmental Science and Pollution Research 25 (5), 4787-4793. DOI:
10.1007/s11356-017-0872-7
Xie Y., Yang Q., Nelson B.D., and DePierre J.W. (2003): The relationship between liver peroxisome proliferation
and adipose tissue atrophy induced by peroxisome proliferator exposure and withdrawal in mice. Biochemical
Pharmacology 66 (5), 749-756. DOI: 10.1016/S0006-2952(03)00386-1
Yahia D., El-Nasser M.A., Abedel-Latif M., Tsukuba C., Yoshida M., Sato I., and Tsuda S. (2010): Effects of
perfluorooctanoic acid (PFOA) exposure to pregnant mice on reproduction. J Toxicol Sci 35 (4), 527-533. DOI:
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Yang Q., Abedi-Valugerdi M., Xie Y., Zhao X.Y., Moller G., Nelson B.D., and DePierre J.W. (2002a): Potent
suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome
proliferator, perfluorooctanoic acid. International Immunopharmacology 2 (2-3), 389-397. DOI: Doi
10.1016/S1567-5769(01)00164-3
Yang Q., Xie Y., Alexson S.E., Nelson B.D., and DePierre J.W. (2002b): Involvement of the peroxisome
proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice.
Biochemical Pharmacology 63 (10), 1893-1900. DOI: 10.1016/s0006-2952(02)00923-1
Yang Q., Xie Y., and Depierre J.W. (2000): Effects of peroxisome proliferators on the thymus and spleen of mice.
Clin Exp Immunol 122 (2), 219-226. DOI: 10.1046/j.1365-2249.2000.01367.x
Yang Q., Xie Y., Eriksson A.M., Nelson B.D., and DePierre J.W. (2001): Further evidence for the involvement of
inhibition of cell proliferation and development in thymic and splenic atrophy induced by the peroxisome
proliferator perfluoroctanoic acid in mice. Biochemical Pharmacology 62 (8), 1133-1140. DOI: Doi
10.1016/S0006-2952(01)00752-3
Zhang H., Ding L., Fang X., Shi Z., Zhang Y., Chen H., Yan X., and Dai J. (2011): Biological responses to
perfluorododecanoic acid exposure in rat kidneys as determined by integrated proteomic and metabonomic
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Zhang H., Hou J., Cui R., Guo X., Shi Z., Yang F., and Dai J. (2013): Phosphoproteome analysis reveals an
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Zheng F., Sheng N., Zhang H.X., Yan S.M., Zhang J.H., and Wang J.S. (2017): Perfluorooctanoic acid exposure
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10.1016/j.taap.2017.09.019

17

6. Annex 1: Toxicokinetic studies
Work in progress and to be added later.
Table 1: Toxicokinetic studies
Substance study design, species, route of
Observed effects
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
to guideline/non-guideline)
Remarks
addressed
(time of sampling)
bw/d) bw/d)
“PFCAs
Summary
In the past decades, a limited set of data was published on the toxicokinetics of PFCAs other than PFOA, such as PFBA, PFHxA, PFHpA, PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA and from EFSA
PFTeDA. The most extensive studies have been carried out in rodents. Oral exposure, mainly by gavage, of experimental animals to PFCAs having a perfluorinated carbon chain Opinion
length of 3–11 was shown to result in an estimated absorption fraction greater than 95% of the administered dose (ATSDR, 2018). None of the experimental studies observed the (2020)
formation of metabolites, suggesting, as previously reported for PFOA (EFSA CONTAM Panel, 2018), that the biotransformation of PFCAs is unlikely in mammals, irrespective of
their chain length.
Although distribution of PFCAs shows species and sex differences, which are attributed, at least in part, to differences in elimination kinetics, blood, liver and kidney are the
tissues exhibiting the highest concentrations of absorbed PFCAs. In blood, PFCAs were found to bind to serum albumin, the affinity generally increasing with PFCAs
hydrophobicity, but decreasing for perfluorinated carbon chain length beyond eight carbons.
The primary route of elimination of PFCAs having a carbon chain length below 10 is via urine, whereas for PFDA, PFUnDA, PFDoDA, PFTrDA and PFTeDA, faecal excretion is
predominant. In rodents, half-lives may vary from few hours (PFBA, PFHxA) to more than 1 month (PFNA, PFDA). Elimination of PFCAs exhibits pronounced sex differences in rats,
with faster elimination in females than in males.
It was shown that transport proteins such as serum albumin, liver fatty acid-binding proteins (L-FABP) and organic anion transporters play a key role in PFCA excretion and/or
reabsorption
(Appendix C).”

18

Commented [A19]: Information on the routes of
take up as the toxicokinteic part is stillin
progress.
Potencies of different chain lengths might be due
to longer time in the body or differences in take
up of the body. This needs to be checked once
the toxicokinetic analysis (for different chain
length, but also differences across species or
sexes) is finished.
Commented [A20R19]: See comment in chapter 1,
no need to summarize individual studies here

7. Annex 2: Repeated dose toxicity studies
Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Observed effects
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)

Perfluoroalkyl carboxylic acids (PFCAs)
TFAA: Trifluoroacetic acid (Syn: Perfluoroethanoic acid, CAS no: 76-05-1, EC no: 200-929-3, Mol. formula: C2HF3O2, MW: 114.023; full and intermediate registration under REACH: 1000-10,000 t/y, harmonised
classification: Skin Corr. 1A, Acute Tox. 4 *, Aquatic Chronic 3)
TFAA (99%)

Body weight ↓ (≈ –17% in HD males)
Liver:
• Abs. + rel. liver weight ↑ (≈ up to 33% in HD)
• Hepatocellular hypertrophy ↑ (in up to 100% of HD rats)
n/sex/group = 10 (m+f)
Haematopoietic system:
• Haemoglobin ↓ (females only, ≈ –8% in HD)
• Mean corpuscular volume ↓ (females only, ≈ –6% in HD)
Exposure: diet
Clinical chemistry:
• Billirubin ↓ (≈ up to −81% in HD)
Doses: 0, 160, 1600, 16000 ppm (0, 9.9,• Glucose ↓ (≈ up to −29% in HD)
98, 1043 mg/kg bw/d for males; 0,
• ALT, ALP ↑ (males only, (≈ up to + 95% and 38% in HD)
12.2, 123, 1216 mg/kg bw/d for
Other effects:
females)
• Urinary ketones ↑ (in up to 100% of male rats)
92-day RDT study (OECD TG 408)

Rat (Wistar)

98/1216

1043/−

9.9/12.2
9.9/123

98/123
98/1216

•

(Anonymo
us, 2016;
BayerCrop
Science,
2014)

1043/12.2 −/123
1043/123 −/1216
9.9/12.2
9.9/12.2
98/1216

98/123
98/123
1043/−

9.9/123

98/1216

PFPA: Pentafluoropropionic acid (Syn: Perfluoroproprionic acid, CAS no: 422-64-0, EC no: 207-021-6, Mol. formula: C3HF5O2, MW: 164.03; pre-registration process under REACH, self classification available)
PFPA

•

PFBA: Perfluorobutanoic acid (Syn: Heptafluorobutyric acid, CAS no: 375-22-4, EC no: 206-786-3, Mol. formula: C4HF7O2, MW: 214,xx?; pre-registration process under REACH, self classification available)
14-day RDT study, non-guideline study Liver:
PFBA,
perfluorobuty to assess liver toxicity
• Catalase activity ↑ (marker enzyme of peroxisomes, ≈ +42%)
−
rate (purity Rat (SD)
• Induction of 80K-protein (associated with peroxisome proliferation) −
not specified),
C3F7CO2
n/sex/group = 3 (m)
Exposure: oral (diet, 0.02%)
Doses: 0, 20 mg/kg bw/d
10-day RDT study, non-guideline study Body weight ↑ (≈ +16%, but it was already significantly higher before
PFBA,
perfluorobuty to assess liver toxicity
treatment)
rate (98%), Mouse (C57BL/6N)
Body weight gain ↑ (≈ +47% (+4.35% in Control, ≈ +6.38% in PFBA
treatment, but no statistical analysis possible))
C3F7CO2
Liver
n/sex/group = 4 (m)
• Abs.+rel. liver weight ↑ (≈ +63% (abs.) or +38% (rel.))
• Hepatic mitochondrial protein content ↑ (≈ +202%)

19

• Body weight,

20
20

kidney, nervous
system, immune
system,
haematopoietic
system,
endocrine
system, clinical
chemistry
• Kidney, nervous
system, immune
system,
haematopoietic
system,
endocrine
system, clinical

(Ikeda et
al., 1985)

(Permadi
et al.,
1992;
Permadi et
al., 1993)

Commented [A21]: We thought it was agreed to
have one table for all studies, not to divide
between repeat tox and developmental tox.
Some studies are extended and cover both. We
need to agree
Commented [A22]: How important is it to fill in
this? Most relevant for guideline studies
deviating form guideline?
Commented [A23]: Too much information for this
table for each substance

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
chemistry

• Hepatic microsomal protein content ↑ (≈ +79%)

Exposure: oral (diet, 0.02%)

• Hepatic peroxisomal catalase specific/total activity ↓↑ (≈ –

37%/+90%)
• Hepatic lauroyl-CoA oxidase specific/total activity ↑

Doses: 0, 24 (ATSDR: 78) mg/kg bw/d

(≈ +112%/+543%)
• Hepatic palmitoyl-CoA oxidation specific/total activity (↑)

(≈ +112%/+539% (but ns))
• Microsomal cytochrome P450 reductase ↑ (≈ +101%)
• Hepatic cytosolic SOD ↑ (≈ +74%)
• Hepatic cytosolic epoxide hydrolase ↑ (≈ +112%)

18-day-ReproTox study (GD1-GD17)
PFBA
(ammonium Mouse (CD-1)
salt, >98%)
n/sex/group = 7-13 pregnant, 2-6
nonpregnant, 30-34 postweaning
Exposure: oral (gavage)

Remark: Perfluoroacetic acid was also tested and did not reveal any
effects (“PFAA was inactive”).
Body weight (ns)
Liver
• Abs. liver weight (pregnant/nonpregnant/postweaning) ↑
35
(≈ +24%/+33%/ns at LOEL)
• Rel. liver weight (pregnant/nonpregnant/postweaning) ↑
35
(≈ +31%/+40%/ns at LOEL)
Remark: Doses were calculated to match PFOA exposure of 1, 5, and
10 mg/kg (Lau et al. 2005). Elimination half-life of PFBA is about 3h
(Chang et al.), PFOA 15-20 days (Lau et al. 2005)

Doses: 0, 35, 175, 350 mg/kg bw/d

•

175
175

Serum concentrations of PFBA (Das et al.,
in pregnant dams (µg/ml):
2008)
Control: 0.002 ± 0.001
35 mg/kg: 3.78 ± 1.01
175 mg/kg: 4.44 ± 0.65
350 mg/kg: 2.49 ± 0.60
Serum concentrations of PFBA
in nonpregnant females
(µg/ml):
Control: 0.006 ± 0.003
35 mg/kg: 1.96 ± 1.0
175 mg/kg: 2.41 ± 1.65
350 mg/kg: 2.67 ± 1.2
Liver concentrations of PFBA
in pregnant dams (µg/g):
Control: 0.003 ± 0.002
35 mg/kg: 1.41 ± 0.42
175 mg/kg: 1.60 ± 0.25
350 mg/kg: 0.96 ± 0.18
Liver concentrations of PFBA
in nonpregnant females
(µg/g):
Control: 0.038 ± 0.017
35 mg/kg: 0.51 ± 0.20
175 mg/kg: 0.86 ± 0.55
350 mg/kg: 0.89 ± 0.38

20

Commented [A22]: How important is it to fill in
this? Most relevant for guideline studies
deviating form guideline?

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
(Data also available for
postweaning stages PD1 and
PD10)
28-day RDT study, non-guideline study Liver:
PFBA,
perfluorobuty to address liver toxicity and its MoA
• Rel. liver weight ↑ (≈ +50% in LD and ≈ +100% in HD of wild type
−
rate (purity Mouse (SV/129 wild-type, PPAR-α null, mice)
not specified), humanized PPAR- α transgenic mouse • Hepatocellular focal necrosis with inflammatory cell infiltrate ↑ (no 35
statistical significance indicated but dose-dependent increase of
C3F7CO2
model)
incidences of mild cases)
• Hepatocyte hypertrophy ↑ (no statistical significance indicated but −
n/sex/group = 10 (m)
−
100% incidence in all dose groups and 0% in C)
• Hepatic replicative DNA synthesis ↑ (only in LD)
−
Exposure: oral (gavage), vehicle: water • mRNA of Cyp4A10/Aco ↑
Remarks: PFBA causes hepatomegaly and hepatocyte hypertrophy
mediated via peroxisome proliferator-activated receptor-α (PPARα)
28-day RDT study, similar to OECD TG Liver:
PFBA,
ammonium 407
• Abs. liver weight (m) ↑ (≈ +27% in MD, no effect after recovery)
6
perfluorobuty Rat (SD)
• Hepatocyte hypertrophy ↑ (no significance indicated but 60%
rate (purity
30
incidence in HD and 0% in C and other dose groups)
not specified),
• Hepatic Acox, Cyp4A1, Cyp2B2, Malic (all m only) ↑
6
n/sex/group = 10 (m+f)
Thyroid:
NH4C3F7CO2
• Follicular hypertrophy/hyperplasia ↑ (no significance indicated but
6
90% incidence in MD and 30% in C)
Exposure: oral (gavage)
Endocrine system:
• Serum total T4 (m) ↓ (≈ −59% in LD)
−
Doses: 0, 6, 30, 150 mg/kg bw/d
• Serum free T4 (m) ↓ (≈ −46% in LD)
−
Clinical chemistry:
• Serum cholesterol (m) ↓ (≈ −20% in MD)
6

• Kidney, liver,

35
175

35
35
35

At 35 mg/kg bw per day (24h (Foreman
et al.,
nervous system, after final dosing)
immune system, Serum: 80 μg/mL
2009)
haematopoietic
Liver: 27 μg/g
system,
endocrine
system, clinical
chemistry

Doses: 0, 35, 175, 350 mg/kg bw/d

Remarks: During recovery, liver weight, histological, and cholesterol
effects were resolved; mRNA transcript data for Acox and Cyp4A1
indicate activation of PPARα
PFBA (purity 90-day RDT study, similar to OECD TG Liver:
not specified), 408
• Abs. liver weight (m) ↑ (≈ +23% in HD)
6
• Hepatocyte hypertrophy ↑ (no significance indicated but 90%
NH4C3F7CO2 Rat (SD)
6
incidence in HD and 0% in C and other dose groups)
• Hepatic Acox, Cyp4A1, Cyp2B2, Malic (all m only) ↑
6
n/sex/group = 10 (m+f)
Thyroid:
• Thyroid: Follicular hypertrophy/hyperplasia ↑ (no significance

21

• Immune system

30
150
30

At 6 mg/kg bw/day in males
Serum: 24.7 ± 17.6 μg/mL
Liver: 7.5 ± 4.5 μg/g

(van
Otterdijk,
2007a)

At 30 mg/kg bw/day in males
(Butenhoff
Serum: 38.04 ± 23.2 μg/mL
et al.,
Liver: 17.4 ± 8.2 μg/g
2012a)

30
6
6
30

• Immune system

30
30
30

At 6 mg/kg bw per day in
males
Serum: 13.6 ± 9.1 µg/mL

(van
Otterdijk,
2007b)

Liver: 3.1 ± 2 µg/g
At 30 mg/kg bw per day in
males

(Butenhoff
et al.,

Commented [A22]: How important is it to fill in
this? Most relevant for guideline studies
deviating form guideline?

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the t,
Observed effects
Substance study design, species, route of
(%), EC/CAS, exposure, doses (guideline/similar
formula
to guideline/non-guideline)
Remarks
Exposure: oral (gavage)
indicated but 90% incidence in HD and 40% in C)
Endocrine system:
• Serum total T4 (m) st, (. —39% in LD)
Doses: 0, 1.2, 6, 30 mg/kg bw/d

PFBA

5-day RDT study
Rat

NO(A)EL
(mg/kg
bw/d)
6

LO(A)EL
(mg/kg
bw/d)
30

6

30

Remarks: During recovery, liver weight, histological, and cholesterol
effects were resolved; mRNA transcript data for Acox and Cyp4A1
indicate activation of PPARa
No effect on gross or microscopic morphology of brain or spinal
cord
Tbc

Key parameters Serum/tissue concentrate- Reference
/ targets not
ion of PFAS /metabolites
addressed
(time of sampling)
Serum: 52.2 ± 25 µg/mL
2012a)
Liver: 16.1 ± 9.1 µgig

•

3M 2007a

Dose: up to 184 mg/kg bw/d
PFPeA: Perfluoropentanoic acid (Syn: Perfluorovaleric acid, CAS no: 2706-90-3, EC no: 220-300-7, Mol. formula: C5HF9O2, MW: 264.05; pre-registration process under REACH, self classification available)

PFHxA: Perfluorohexanoic acid (Syn: Undecafluorohexanoic acid, CAS no: 307-24-4, EC no: 206-196-6, Mol. formula: C6HFu.O2, MW: 264,xx?; under PBT assessment under REACH, self classification available)
PFHxA (purity 5-day RDT study, non-guideline study Body weight (m/f) (ns)
Liver:
not specified) Mouse (ddY)
• Rel. liver weight '1' (m: ns; f, =+17% at LOEL)
• Peroxisomal 13-oxidation l'
n/sex/group = 3-5 (m+f)

• Kidney, nervous
system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Kudo et
al., 2006)

• no
Plasma conc. (ng/ml) at 62.6
histopathological mg/kg bw/d at end of study:
378 ±178 (ml
description for
Thyroid gland
129 ± 16 (f)

(NTP,
2019b)

50

100
50

500

1000

250/250
125/250
250/500
250

500/500
250/M
M

Plasma conc. (ng/ml) at 250
mg/kg bw/d at end of study:

250/500

==

1297 ± 265 (m)

500
250/500

1000
500/1000

—

62.5

Exposure: i.p.
Doses: 0, 50, 100, 150 mg/kg bw/d
PFHxA (>99%) 28-day RDT study, similar to OECD TG Body weight ,I, (m, ,- —16% at LOEL; f ns)
407
Liver:
.Abs. liver weight '1` (m/f, . +27%/+14% at LOEL)
Rat (SD)
• Rel. liver weight 1' (m/f, . +14%/+15% at LOEL)
• Hepatocellular hypertrophy 1' (m, 9 of 10 at 500 mg/kg bw/d)
n/sex/group = 10 (m+f)
.Acyl-CoA oxidase 1' 1.-. +141% in males at 250 mg/kg bw/d)
Kidney:
Exposure: oral (gavage; in 2% Tween 'Rel. kidney weight 1' (m/f, . +12%/+12% at LOEL)
Immune system
80/ deionized water)
• Abs. thymus weight .1, (m, =-27% at LOEL. Rel ns, f not measured)
• Spleen: increased extramedullidary hematopoiesis
Doses: 0, 62.5, 125, 250, 500, 1000
Thyroid weight
mg/kg bw/d
•Thyroid weight changes at all doses (m+f), but ns and not clearly
dose related
22

Liver conc (ng/g) at 250 mg/kg
bw/d at end of study:
655 ± 148 (m)

Exp.no.
C20613

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table\
Substance study design, species, route of
Observed effects
NO(A)EL
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg
formula
to guideline/non-guideline)
bw/d)
Remarks
Haematopoietic system:
• Erythrocytes (=-5%/-7% at LOEL), haemoglobin (=-3%/-6% at
-/125
twice daily at one-half dose (total):
31.3 (62.6), 62.5 (125), 125 (250), 250 LOEL), haematocrit (,... —4%/-7% at LOEL) (m/f) 4,
Endocrine system:
(500), 500 (1000) mg/kg bw/d
.T3 (.. -18% in LD), fT4 (.. -25% in LD), tT4 (. -20% in LD) (m) 4, (f ns)
Clinical chemistry:
.ALT, ALP (m), AST (m+f) '1`
250/250
• Serum cholesterol 4, (m, z.. -20% at LOEL)
Other effects:
• Degeneration and hyperplasia of olfactory epithelium (m+f)
125/125
Reproductive tissues (doses tested: 0, 250, 500,1000 mg/kg bw/d)
• Cauda epididymal sperm counts 4, (m, =-25%)
• Epididymal weight 4, (m,. —5% at 1000 mg/kg bw/d)
• Epididymal histopathology: Exfoliated germ cells (1/10 at LOEL)
500
•Testis weight: in treated animals similar to controls
500
• Spermatid counts: in treated animals to controls
500
• Seminiferous tubule spermatid retention of the testis in 2/10 rats at
1000 mg/kg bw/d and 1/10 control rat (m)
• Estrus cyclicity (f, ns)
PFHxA,
28-day RDT study with reproduction/ *Slash (/) indicates here the changed max. dose in HD
150
Perfluorohexa developmental toxicity screening test, Mortality 1` (death of 5/15 m and 6/15 fin HD)
Body weight 4,
50
noic acid
OECD TG 422
Liver:
(98.5%), CAS: Rat (SD)
•Abs. + rel. liver weight (m/f) Is
50
307-24-4/EC:
206-196-6,
50
C6HFu.O2
n/sex/group = 10-15 (m)
• Hepatocellular hypertrophy '1` (minimal (MD) to mild (HD)
Kidney:
• Papillary necrosist
150
Exposure: oral (gavage), vehicle:
Immune system:
deionized water
• Lymphoid necrosis/depletion of T and B areas of lymph nodes,
150
spleen, thymus I'
Doses: 0, 50,150, 300-450 mg/kg bw/d.Abs. thymus weight (m) 4, (considered spurious by authors)
150
(reduced on day 4 from 450 to 300
Haematopoietic system:
mg/kg bw/d due to lethality in 4/15
. Mean corpuscular haemoglobin (m) 4,
150
males and 4/15 females within the first. Haemoglobin (m),j,
150
4 days of dosing)
.Globulin (m)4,
150
• No changes in total or differential leucocyte counts
Clinical chemistry:
• Serum cholesterol 4, (m; f ns)
150
•Triglycerides (NA)
23

LO(A)EL
(mg/kg
bw/d)

Key parameters Serum/tissue concentrate- Reference
/ targets not
ion of PFAS /metabolites
addressed
(time of sampling)

62.5/250

500/500
62.5

=
1000
1000

300-450
150/300
150/300450
150
300-450
300-450
300-450
300-450
300-450
300-450

300-450

Blood samples taken 1, 2, 4, 8, (Kirkpatrick
24 h on study day 0 and 25
, 2005)
from three rats per group,
Proj. ID:
Urine samples collected from WILall animals 0-6, 6-12, 12-24 h 534001)
following last dosing
(WIL,
Half-life in serum and for
2005)
urinary elimination about 2-3
h
Exposure in male rats 2-4-fold
higher than in female rats
(e.g., AUC (ngxh/mL): Day 25,
HD males: 1637875; Days 25
HD females: 887031)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)

PFHxA
(98.5%)

Thyroid
• No changes in thyroid weights
Endocrine system:
• Hyperplasia of adrenal cortex ↑
Other effects:
• Litter size ↓
• Pup number ↓
• Implantation size (f) ↓
• Food consumption (f) ↓ during gestation and lactation
• Hemorragic ring around iris ↑
90-day RDT study, similar to OECD TG Body weight gain (m) ↓ (≈ −7% in LD&HD, –10% in MD at day 90)
408
Body weight gain (f) (≈ −5% in LD&HD at day 90, but ns)
Liver:
Rat (SD)
• Rel. liver weight (m) ↑ (≈ +22% in HD) (f ns)
• Centrilobular hepatocellular hypertrophy (m)
n/sex/group = 10 (m+f)
• Hepatic peroxisomal β-oxidation (m) ↑
Kidney:
• Rel. kidney weight (m) ↑ (≈ +8% in LD)
Exposure: oral (gavage)
Immune system
• White blood cell count (ns)
Doses: 0, 10, 50, 200 mg/kg bw/d
• Thymus and spleen weight (m+f) (ns)
Haematopoietic system:
• Erythrocytes, haemoglobin ↓ (≈ −8% in HD)
• Reticulocyte ↑ (≈ +59% in HD)
• Globulin (m)↓ (≈ −8% in HD)
Clinical chemistry:
• Serum cholesterol (m) ↓ (≈ −26% in MD) (f ns
• Triglycerides not measured
• Calcium (m) ↓ (≈ −3% in MD)
• Serum ALT (≈ +237% in HD) and ALP (m) ↑ (≈ +109% in HD)

150

300-450

150
150
150
150
10

300
300
300
300
50
50

50
50
50

200
200
200

−

10

50
50
50

200
200
200

10

50

10
50

50
200

Remark: “Based on liver histopathology and liver weight changes, the
no-observed-adverse-effect level (NOAEL) for oral administration was
50 mg/kg bw/day for males and 200 mg/kg bw/day for females.” (200
mg/kg bw/d is an unbounded NOAEL,i.e. the NOAEL is the highest
measured dose and the “real” NOAEL could be even higher)
PFHxA
90-day RDT study, OECD TG 408, plus Body weight (m) ↓ (≈–10% at day 90 at LOEL)
100
(sodium salt, 30 day recovery group
Liver:
100%)
• Abs./rel. liver weight ↑ (≈ +63% in HD)
100
Rat (Crl:CD(SD))
• Hepatocellular hypertrophy ↑ (no significance indicated but for
20/100
males 40% incidence in MD, 100% in HD and 0% in C and LD)

24

500
500
100/500

(Chengelis
et al.,
2009)

•

(Loveless
et al.,
2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
n/sex/group = 10 (m+f)
Exposure: oral (gavage)
Doses: 0, 20, 100, 500 mg/kg bw/d

PFHxA
sodium salt
(100%)

• Hepatic peroxisomal β-oxidation (m/f) ↑ (≈ +85% in MD, ≈ +330% in 20/100

HD)
Kidney:
• Rel. kidney weight ↑ (≈ +16% in HD)
100
• Urine volume ↑ (≈ +207% in HD)
100
Immune system
• Abs. thymus weight (m) ↓ (≈ –31% at LOEL, rel. weight ns)
100
• Abs. spleen weight (m) ↓ (≈ –16% at LOEL rel. weight ns)
100
Haematopoietic system:
• Erythrocytes (≈ −31% in HD) , haemoglobin (≈ −36% in HD) ↓
100
• Reticulocyte ↑ (≈ +210% in HD)
100
Thyroid:
• Thyroid hypertrophy ↑ (no significance indicated but for females
100
40% incidence in HD, 0% in other dose groups)
• Thyroid weight ↑ (only in f after 30 day recovery)
100
Clinical chemistry:
• Serum ALT (m) ↑ (≈ +133% in LD)
−
• Cholesterol (m) ↓ (≈ –35% at 100 mg/kg bw/d, at HD only
significant after 3 months of treatment, effect not considered
adverse)
• Triglycerides (ns)
Other effects:
20
• Degeneration/atrophy in nasal cavity (no significance indicated but
for males 40% incidence in MD, 70% in HD and 0% in C and LD)

Remark: “subchronic toxicity no observed adverse effect level
(NOAEL)was 20 mg/(kg day), based on nasal lesions observed at 100
and 500 mg/(kg day)”
110-day RDT study, as part of a one- Body weight (m) ↓ (≈ –12% at LOEL)
20
generation reproduction study, OECD Body weight (f, GD0-7)) ↓ (≈ –31% at LOEL)
100
TG 415
Rat (Crl:CD(SD))
n/sex/group = 10 (m+f)
Exposure: oral (gavage)
Doses: 0, 20, 100, 500 mg/kg bw/d

25

100/500

500
500
500
500
500
500
500
500
20

100

100
500

• Liver, kidney,

nervous system,
immune system,
haematopoietic
system, thyroid,
endocrine system

(Loveless
et al.,
2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFHxA
ammonium
salt (93.4%)

Combined Developmental and
perinatal/postnatal reproduction
toxicity study

Phase I
Body weight (ns)
Liver:
ICH Harmonised Tripartite Guideline - • Abs/rel liver weight (ns)
S5(R2), stage C-F
Phase II
Mouse Crl:CD1(ICR), 69-76 d.o. at
Body weight (slight reduction at mid and high dose on GD 18,
mating
lowest dose slightly higher than control)
Liver:
• Abs/rel liver weight (ns)
F0: 20 F/dose GDs 6-18(Males only
used for breeding, no treatment)

• Postnatal

functional
parameters are
not evaluated in
this type of study

(Iwai and
Hoberman,
2014; Iwai
et al.,
2019)
Study
reports:
Hoberman,
2011a
(Phase I)
Hoberman,
2011b
(Phase II)

F1: 20/sex/dose (no direct treatment;
Remark: Iwai and Hoberman 2014 abstract: “Based on these data,
observed until weaning to sexual
the maternal and reproductive no observable adverse effect level of
maturity)
PFHxA Ammonium Salt is 100 mg/kg bw/d.”
Exposure: oral (gavage; in deionized
water)
Phase I:
Doses: 0, 100, 350, 500 mg/kg bw/d
Phase II
Doses: 0, 7, 35, 175 mg/kg bw/d
PFHxA
(98.1%)

104-weeks Carcinogenic study
Combined chronic tox/carc study
Rat Crl:CD(SD)
n/sex/group = 60-70
Male Dosage: 0, 2.5, 15, 100 mg/kg
bw/d
Female Dosage: 0, 5, 30, 200 mg/kg
bw/d

Mortality ↑ (f only: 22% survival at 200 mg/kg bw/d vs 36% survival
in control, i.e. +100% mortality at 200 mg/kg bw/d, but no statistical
testing)
Body weight (no effect)
Liver:
• Hepatocellular necrosis (weak increase at highest dose in m+f)
Kidney:
• Necrosis ↑ (f only, minimal to severe papillary necrosis in 17 of 70 30
test animals and/or minimal to moderate renal tubular
degeneration in 7 of 70 test animals)
• Urinary parameters: mean urine volume ↑ (f); slightly lower specific 30
gravity at 200 mg/kg bw/d ↓ (f, 26. Week)
Haematopoietic system:
• White blood cell count (ns)
• Red blood cell count ↓ (–8% after 51 weeks only in females)
30
• Heamoglobin ↓ (–5% after 51 weeks only in females)
30
Clinical chemistry:

26

(Klaunig et
al., 2015)

200

200

200
200

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Serum triglycerides ↓ (m: –43% at2.5 and 100, but not at 15 mg/kg –

2.5

bw/d after 52 weeks, authors conclude no effect)
• Serum LDL (f: –44% at LOEL after 26 weeks, but not after 52 weeks)

30

200

• Serum HDL (ns)

Remark: not tumorgenic (f+m) all dosages, authors in abstract:
“heamatology and serum chemistry unaffected”, no statistically sign.
Alterations in hormones LH, Testosterone, Estradiol (Jung et al. 2016
stated: there were no histological changes in ED-related tissues)
PFHpA: Perfluoroheptanoic acid (Syn: Tridecafluoroheptanoic Acid, CAS no: 375-85-9, EC no: 206-798-9, Mol. formula: C7HF13O2, MW: 364,xx?; pre-registration process under REACH, self classification
available)
5-day RDT study, non-guideline study, Liver:
PFHpA,
Perfluorohept to assess hepatic peroxisomal β• hepatic peroxisomal β-oxidation ↑ (m/f)
30/160
oxidation through PFCAs
anoic acid
(purity not
Remarks: potency of the induction of peroxisomal β-oxidation was
Rat (Wistar)
specified),
compared between PFCAs; highly significant correlation between the
n/sex/group = 4 (m+f)
CAS: 375-85-9
induction and hepatic concentrations of PFCAs in the liver regardless
/ EC: 206-798of their carbon chain lengths
9, C7HF13O2 Exposure: i.p.

• kidney, nervous

160/−

concentrations of PFHA in the (Kudo et
system, immune liver were below detection
al., 2000)
limit <3 g:g liver
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

Doses: 0, 30, 160 mg/kg bw/d
PFHpA (purity 5-day RDT study, non-guideline study, Liver:
not specified) Mouse (ddY)
• Abs. and rel. liver weight ↑ (m/f, ≈ +68% in MD)
20/50
• Peroxisomal β-oxidation ↑
−/20
• Activity of hepatic microsomal 1-Acyl-GPC acyltransferase (m, only
n/sex/group = 3-5
−
HD tested) ↑
Exposure: i.p.
Doses: 0, 20, 50, 100 mg/kg bw/d

100

Remarks: the longer the perfluoroalkyl chain, the more PFCA
accumulates in the liver; accumulated PFCAs induce hepatomegaly,
peroxisomal β-oxidation and microsomal 1-acyl-GPC acyltransferase

PFHpA (purity 3-day RDT study, non-guideline study Liver:
not specified) Mouse (C57BL/6)
• Hepatic mRNA levels of Cyp4a10 ↑
n/sex/group: 4

• kidney, nervous

50/100
20/50

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

• kidney, nervous

−

Remarks: Mechanistic study to assess the MoA of PFCAs

Exposure: i.p.

27

20

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Kudo et
al., 2006)

(Abe et al.,
2017)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0 , 20 mg/kg bw/d
Liver:
combined 90-day RDT study with
PFHpA as
reproduction/developmental toxicity • Centrilobular hypertrophy of hepatocytes ↑
Sodium
perfluorohept screening (similar to OECD TG 408 and • Abs. and rel. liver weight ↑
anoate
Clinical chemistry:
422)
(>99.3%)
• ALT levels in lactating females (D21) ↑
Mouse (CD1)
• ALP, ALT and Trig. in m and in ALP and Trig. in non-mated f ↑
exposure time F0, m: 90d prior to
Endocrine system:
mating (total 109-113 d); F0, f: 90d
• Thyroid T4 levels in serum ↓ (m only)
prior to pairing and until lactation
(total exposure time: 130-142 d)
Developmental effects in F1:
exposure time F1: during PND 22 to 42 • Centrilobular hypertrophy of hepatocytes ↑
(total of 21 days)
• Hepatocellular necrosis (single cell to coalescing) ↑
• Abs. and rel. liver weight ↑
n/sex/group: in F0 20 (except 25 f for • T4 serum levels in f ↑

(Anonymo
us, 2017)

•

−/−
0.5/0.5

0.5/0.5
10/10

0.5
10/10

10
50/50

0.5

10

−/−
0.5/0.5
0.5/10
−

0.5/0.5
10/10
10/50
0.5

controls and highest dose and 15 for
clinical pathology phase); in F1 n=16-20• Cleft palate in 6 pups from 1 litter at LD
• Cleft palates in 3 pups from 2 litters at HD
• % of males per litter ↓ in LD
Exposure: oral (gavage), vehicle:
• Postnatal survival ↓ in HD
deionised water
• Mean body weight ↓ in HD
• Vaginal patency ↑ in HD
• Adrenal rel. and abs. weights in f ↑ in HD
Doses: 0, 0.5, 10, 50 mg/kg bw/d
PFOA: Perfluorooctanoic acid (Syn: Pentadecafluorooctanoic acid, CAS no: 335-67-1, EC no: 206-397-9, Mol. formula: C8HF15O2, MW: 414,xx?; registered under REACH, self classification available)
PFOA,
5-day RDT study, non-guideline study
Perfluoroocta Rat (Wistar)
noic acid
(analytical
n/sex/group = 4
grade), EC:
206-397-9,
CAS: 335-67- Exposure: i.p. injection (vehicle:
1, C8HF15O2 propyleneglycol:water (1:1, v:v)

Liver:
• Peroxisomal β-oxidation ↑ (m/f)

• kidney, nervous

5/20

Remarks: Mechanistic study to assess the MoA and to investigate
influence of chain length; peroxisomal β-oxidation is statistically
highly correlated with PFCA concentration in the liver (r=0.850,
p<0.001) à internal dose in liver decisive for effect, not carbon chain
length or sex

Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d
PFCAs assessed: PFHpA, PFOA, PFNA,

28

10/−

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Kudo et
al., 2000)

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Formatted: French (France)

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFDA
PFOA (purity 5-day RDT study, non-guideline study Liver:
• Abs. and rel. liver weight (m/f) ↑ (≈ +39% in LD)
−/−
not specified) Mouse (ddY)
• Peroxisomal β-oxidation ↑
−/−
• Activity of hepatic microsomal 1-Acly-GPC acyltransferase (m, only
n/sex/group = 3-5
20 mg/kg bw/d tested) ↑
−
Exposure: i.p.
Doses: 0, 2.5, 5, 10, 20 mg/kg bw/d

20

Remarks: “(…) the longer the perfluoroalkyl chain, the more PFCA
accumulates in the liver (… and) accumulated PFCAs induce
hepatomegaly, peroxisomal β-oxidation and microsomal 1-acyl-GPC
acyltransferase (…)”(from abstract; refers to C6-C9)

PFOA (purity 3-day RDT study, non-guideline study Liver:
• Rel. liver weight ↑ (≈ +100%)
not specified) Mouse (C57BL/6)
• Hepatic mRNA levels of Cyp4a10, Acox1, Cyp2b10, Aldh1 ↑
n/sex/group = 4

• kidney, nervous

2.5/2.5
2.5/2.5

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

• kidney, nervous

−
−

20
20

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

−

1

• kidney, nervous

1
−
−
−

5
1
1
1

−
1
−

1
5
1

Remarks: Mechanistic study to assess the MoA of PFCAs

Exposure: i.p.

Chain length (C6-C9
investigated) and sex
dependent accumulation of
PFCAs in the liver (higher
concentrations in males and
with higher C length)

(Kudo et
al., 2006)

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(Abe et al.,
2017)

Doses: 0 , 20 mg/kg bw/d

Formatted: French (France)

PFOA (>96%) 7-days RDT study, non-guideline study Body weight ↓
Liver:
Mouse (Balb/c)
• Abs. liver weight ↑ (≈ +60% at LOAEL)
• Necrosis and vacuolation of hepatocytes ↑
n/sex/group = not reported
• Hepatic triglycerides ↑
• mRNA levels of fatty acid translocase and lipoprotein lipase ↑
Clinical chemistry:
Exposure: oral (vi distilled water)
• ALT ↑ (≈ +80% at LOAEL)
• Serum fatty acids ↓ (≈ -62% at LOAEL)
Doses: 0, 1, 5 mg/kg bw/d
• Serum triglycerides ↓ (≈ -58% at LOAEL)
Remarks: Mechanistic study to assess the MoA of PFCAs

29

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Hui et al.,
2017)

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFOA (purity 14-days RDT study, non-guideline
not specified) study
Rat (SD)
n/sex/group = 7 (m only)
Exposure: oral (gavage)
Doses: 0, 1, 5, 25 mg/kg bw/d
PFOA (96%)

21-days RDT study, non-guideline
study
Mouse ( Balb/c)
n/sex/group = 3-10 (m only)
Exposure: oral (gavage)
Doses: 0, 1.25 mg/kg bw/d

Body weight ↓
Liver:
• Abs. + rel. liver weight ↑
• Activity of hepatic superoxide dismutase and glutathione
peroxidase ↑
• MDA content in liver ↑
• Expression of PPARα mRNA ↑
• Expression of CYP4A1 mRNA ↑
• PPARα protein ↑
Remark: CYP4A1 is a PPARα-related gene
Liver:
• Rel. liver weight ↑ (≈ +120%)
• Glycogen (≈ −80%) and glucose (≈ −33%) content in the liver ↓
• Pyruvate ↑ (≈ +80%)
Clinical chemistry:
• Fasting blood glucose levels ↑ (≈ +50%)
• Blood glucagon ↑ (≈ +50%)

5

25

• kidney, nervous

1

5

−
1
–
–
–

1
5
1
1
1

−
−
−

1.25
1.25
1.25

−
−

1.25
1.25

1
1
1
1
1

5
5
5
5
5

1
1
1
1
1
1

5
5
5
5
5
5

1
−

5
1

• kidney, nervous

1

system, immune
system,

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

PFOA level in liver: 125.9 ±
system, immune 10.0 μg/g
system,
haematopoietic
PFOA level in serum: 55.5 ±
system, thyroid,
0.50 μg/mL
endocrine system

(Wang et
al., 2017)
Article in
Chinese,
only
abstract in
English

• kidney, nervous

(Zheng et
al., 2017)

• kidney, nervous

(Wu et al.,
2018)

Remarks: Mechanistic study to assess the MoA: indicates that
subacute exposure to PFOA might enhance glycogenolysis and
gluconeogenesis and promote carbohydrate consumption

PFOA
21-days RDT study, non-guideline
(ammonium study
salt, <98%) Mouse (Kunming)

Liver:
• Abs. and rel. liver weight ↑
• Hepatic functional enzymes (GPT, GOT) ↑
• Hepatic triglycerides ↑ (≈ +150% in HD)
• Visible vacuoles around liver portal area ↑
n/sex/group: 8 (m only)
• Hepatic FGF21 protein ↑
Clinical chemistry:
Exposure: oral (gavage, in peanut oil + • Serum cholesterol ↓ (≈ −26% in MD)
• Serum glucose ↓ (≈ −25% in HD)
0.5% DMSO)
• Serum insulin ↓ (≈ −45% in HD)
• Serum triglyceride ↓ (≈ −57% in HD)
Doses: 0, 1, 5 mg/kg bw/d
• Serum L-LDL ↑ (≈ +140% in HD)
• Serum H-LDL ↓ (≈ −50% in HD)
Other effects:
• Insulin-positive cells in pancreatic islets ↑
PFOA
2, 8 or 16 weeks RDT study, nonBody weight ↓ (≈ −22 – −37%)
(ammonium guideline study
Liver:
• Abs./rel. liver weight ↑ (≈ +50%)
salt, >98%) Mouse (57BlL/6)

−

30

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Li et al.,
2019)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Replication of hepatocytes (week 2+8) ↑ (4.3-fold increase of the

n/sex/group = 5 (m only)
Exposure: oral (gavage, in distilled
water)

hepatocytes DNA synthesis
• Hepatic peroxisomal ß-oxidation activity (week 2-16)
• Activation of PPARα
• Expression of fatty acid metabolism genes ↑

−
−
−
−

1
1
1
1

340
37/115

1600
115/340

–/37

–/115

115/–

340/–

760
240
13/51
13
13
13/–

1900
760
51/240
51
51
51/13

–
13/–
–
–
–

13
240/13
13
13
13

haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

Remarks: Mechanistic study to assess the MoA
Doses: 0, 1 mg/kg bw/d
PFOA

28-day RDT study

(ammonium Rat (ChR-CD albino)
salt, purity
not specified)
n/sex/group = 5
Exposure: oral (dietary)

Mortality 100% within 7 days
Body weight ↓ (m, ≈ –21/–26.5/–29/–33%; f, -22.5/-23/-18/-20%
after 1/2/3/4 week at LOEL)
Liver:
• Abs. liver weight ↑ (m, ≈ +38% at 30 ppm, ≈ +60% at 300 ppm; f:
≈ +25% at LOEL)
• Rel. liver weight ↑ (m, ≈ +104% at LOEL; f: ns)

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

Remarks: Acute oral toxicity (LD50 540 mg/kg), primary skin irritation
(no effects), eye irrigation (moderate) and one-hour inhalation data
(no death from inhalation) as well as in vitro mutagenicity tests (all
(equivalent to 0, 3.5, 12, 37, 115, 340, negative) are also available
“1600”, “4800” mg/kg bw/d, last two 3M’s effort
values hypothetical as animals in these
groups died, female doses slightly
higher than males)

(Griffith
and Long,
1980;
Study
report by
Metrick
and
Marisa,
1977)

Doses: 0, 30, 100, 300, 1000, 3000,
10000, 30000 ppm

PFOA (“FC- 28-day RDT study
143” (3M), Mouse (albino)
ammonium
salt, purity
not specified) n/sex/group = 5
Exposure: oral (dietary)
Doses: 0, 30, 100, 300, 1000, 3000,
10000, 30000 ppm (of diet)
(equivalent to 0, 13, 51, 240, “760”,
“1900”, “6200”, “19000” mg/kg bw/d,
last four values hypothetical as animals
in these groups died, female doses

Mortality 100% after 9 days
Mortality 90% after 26 days
Body weight (week 1) ↓ (m/f, –18%/-29% for m/f at LOEL)
Body weight (week 2) ↓ (-32%/-28% for m/f at LOEL)
Body weight (week 3) ↓ (-34%/-33% for m/f at LOEL)
Body weight (week 4) ↓ (-37.5%/-25% for m/f at LOEL)
Liver:
• Abs. liver weight ↑ (m/f, ≈ +171%/+202% at LOEL)
• Rel. liver weight ↑ (m/f, ≈ +252%/+294% at LOEL)
• Hepatic hypertrophy ↑ (mild to moderate)
• Hepatic lipid vacuolation ↑ (minimal to mild)
• Hepatocellular degeneration or necrosis ↑ (minimal to mild)

31

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Study
report by
Christophe
r and
Marias,
1977;
Griffith and
Long,
1980)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
slightly higher than males)
PFOA

90-day RDT study

(ammonium Rat (ChR-CD albino),
salt)
n/sex/group = 5
Exposure: oral (dietary)

Body weight ↓ (m, ≈ –12% at LOAEL)
Liver:
• Abs. liver weight ↑ (m, ≈ +51% at LOAEL)
• Rel. liver weight ↑ (m/f, ≈ +62/+28% at LOEL)
Kidney:
• Abs. kidney weight ↑ (m, ≈ +24% only at 30 ppm)
• Rel. kidney weight ↑ (m, ≈ +22% at LOEL; f, +30% only at 10 ppm)

22

75

7.2/–
7.2/22

22–
22/75

7.2/–

7.2/–

• nervous system,

immune system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(equivalent to 0.7, 2.2, 7.2, 22, 75
mg/kg bw/d, female doses slightly
higher than males)
90-day RDT study

(ammonium Rhesus monkey,
salt)

0 ppm: ND/ND
10 ppm: 21/NM
30 ppm: 34/0.15
100 ppm: 36/NM
300 ppm: 38/0.25
1000 ppm: 49/0.65

(Study
report by
Goldenthal
, 1978a;
Griffith and
Long,
1980)

ND = None detected
NM = Not measured

Doses: 0, 10, 30, 100, 300, 1000 ppm

PFOA

Fluorine in pooled (n=4-5)
serum (ppm) (m/f)

Mortality:
• 30 mg/kg bw/d: 2 f and 1 m died in weeks 7-12
• 100 mg/kg bw/d: All died in weeks 2-5

10

30

• body weight,

liver, kidney,
nervous system,
immune system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

n/sex/group = 2
Exposure: oral (gavage)
Doses: 0, 3, 10, 30, 100 mg/kg bw/d

Ammonium PFOA in serum
(ppm) (m/f)

(Study
report by
Goldenthal
0 mg/kg bw/d: NM/1
3 mg/kg bw/d: 48.53/50.65 , 1978b;
10 mg/kg bw/d: 45.71/71.79 Griffith and
Long,
30 mg/kg bw/d: 145/NM
1980)
100 mg/kg bw/d: NM/NM
Ammonium PFOA in liver
(ppm) (m/f):
0 mg/kg bw/d: 0.05/0.07
3 mg/kg bw/d: 3/7
10 mg/kg bw/d: 9/10
30 mg/kg bw/d:
60.125/80.125
100 mg/kg bw/d: 100/325

PFOA
13-week (91 days)+8week recovery
(ammonium Rat (Crl:CD®BR)
salt)
n/sex/group = 10-25 (males only)
Exposure: oral (dietary)

Body weight (ns)
Liver:
• Abs. liver weight ↑ (≈ +36%% at LOEL in week 8)
• Rel. liver weight ↑ (≈ +37% at LOEL in week 8)
• Hepatocellular hypertrophy ↑
• Hepatic palmitoyl CoA oxidase activity ↑ (≈ +133% at LOEL after in
week 14)

32

• kidney, nervous

0.06
0.06
0.06
0.65

0.65
0.65
0.65
1.94

system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Palazzolo,
1993)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 0 (pair-fed to 100 ppm), 1,
10, 30, 100 ppm

Remark: Effects seemingly reversible. Overall, NOAEL is 100 ppm (6.5
mg/kg bw/d), NOEL is 1 ppm (0.06 mg/kg bw/d)

equivalent to 0, 0.06, 0.65, 1.94, 6.5
mg/kg bw/day (calculated in study
report)
26 weeks + recovery (2 control and 2
(ammonium mid-dose monkeys) 13 weeks more
salt, 95.2% Cynomolgus monkey
purity)
PFOA

Body weight
• 30 mg/kg bw/d (in first 11 days) -3 to -7.5% (no food consumption), 10
then 10 days of no dose, then continued with 20 mg/kg bw/d with
reduced body weight and body weight gain until week 14, then
steep increase in body weight but overall weight still reduced.
n/sex/group = (males only)
• Recovery animals from 10 mg/kg bw/d group much lower body
weight gain in 14 weeks after treatment
Liver:
Exposure: oral (gelatine capsules)
• Abs. liver weight ↑ (≈ +35% at LOEL)
0
• Rel. liver weight ↑ (≈ +57% at LOEL)
10
Doses: 0, 3, 10, 20-30 mg/kg bw/d (30 Endocrine system:
mg/kg bw/d was reduced to 20 mg/kg • Total T4 ↓ (≈ –37.5/–35/–31% at MD after 5/10/14 weeks; ≈ –33% bw/d at day 22 after no dosing on days at LOEL after 27 weeks)
12-21)
• Free T4 ↓ (≈ –32/–27/–38% after 5/10/27 weeks at LOEL)
3
• Free T3 ↓ (≈ –31/–47/–40% after 5/10/27 weeks at LOEL)
10
Clinical chemistry:
• Triglycerides ↑ (≈ +145/+109% after 5/27 weeks at HD; ≈ +120%
3
after 14 weeks at MD)
• Total bilirubin ↓ (≈ –60% only after 10 weeks, only at MD)

Remarks: Effects are mainly in the 30/20 mg/kg bw/d group, which
was an inconsistent treatment due to toxic effects (no food uptake)
in the first 10 days with the 30 mg/kg bw/d dosage. Data on some
hepatic marker enzymes also available but no methods described…
Liver PFOA content returned to initial levels after 14 weeks of
recovery
PFOA
70-days (parental-generation, before Body weight ↓ (≈ –7%)
1
(ammonium cohabilitation), 2-generation guideline Liver:
• Abs.+rel. liver weight ↑ (≈ +20% at LOEL)
salt, 97.99%) study EPA OPPTS 8703800
Kidney:
• Rel. kidney weight ↑ (≈ +16.5% at LOEL)
Rat (Sprague-Dawley)
n/sex/group = 30 (males only)

33

• kidney, nervous

20-30

system, immune
system,
haematopoietic
system, thyroid

10

Liver tissue PFOA
concentration (µg/g) (values
of individuals, mean ± sd in
bold))

(Butenhoff
et al.,
2002;
Study
Control: 0.09, 0.23, <LOQ (2x) report by
Thomford,
3 mg/kg bw/d: 15.2, 18.5,
2001)
11.3, 18.3 (15.8 ± 3.4)
10 mg/kg bw/d: 21.9, 6.3, 8.9,
18.8 (14.0 ± 7.6)

3
20-30

30/20 mg/kg bw/d: 16, 83.3
3

Control recovery: <LOQ (2x)

10
20-30

10 mg/kg bw/d recovery: 0.15,
0.08

10

3
1
1

• Nervous system,

immune system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Butenhoff
et al.,
2004)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Exposure: oral (gavage)
Doses: 0, 1, 3, 10 mg/kg bw/d
PFOA
Liver:
7 day-study
(ammonium Mouse (PPARα-null mice (129S4/SvJae- • Abs. liver weight ↑ (≈ +30% at LOEL)
salt, purity) Pparatm1Gonz/J) and wildtype
• Rel. liver weight ↑ (≈ +32% at LOEL)
(WT)(129S1/SvlmJ - SV/129 and CD-1)) • Hepatocyte hypertrophy (lesion score) ↑ (≈ +73% at LOEL)
• Labelling index (LI) ↑ (≈ +3750% at LOEL)

• Body weight,

0
0
0
3

1
1
1
10

•

n/sex/group = (m only)

• Peroxisome proliferation
• Increased cell proliferation (only in wild-type)

Exposure: oral (gavage), PFOA in
deionized water

• Primary effects of PFOA on WT mice was on genes associated with

kidney, nervous
system, immune
system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

fatty acid metabolism, consistent with activation of PPARα
• Other alterations on genes related to inflammation
• Altered expression of genes associated with cell cycle control,

Control: 0.042 ± 0.020/ 0.046
± 0.056/ 0.053 ± 0.059
1 mg/kg: 58.2 ± 6.7/ 56.9 ±
7.7/ 37.1 ± 7.9
3 mg/kg: 111.1 ± 23.4/ - / 90.7
± 9.2
10 mg/kg: 296.3 ± 57.0/ 310.0
± 59.2/ 209.5 ± 29.1

peroxisome biogenesis and proteasome structure/organization
• Cyp2b and Cyp2c genes upregulated

Remark: “In wild-type mice, PFOA (…) induced changes consistent
with activation of PPARα. (…)In PFOA-treated null mice, changes
were observed in transcripts related to fatty acid metabolism,
inflammation, xenobiotic metabolism, and cell cycle regulation.
Hence, a component of the PFOA response was found to be
independent of PPARα.” (from abstract of Rosen et al. 2008)
PFOA

14-day-study

(ammonium Rat (Crl:CD®(SD)IGS BR)
salt,
linear/branch
n/sex/group = 10 (m only)
ed (80:20)
isoforms)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

Body weight (day 7) ↓ (≈ –17% at LOEL)
Body weight (day 13) ↓ (≈ –19% at LOEL)
Body weight gain (0-13) ↓ (≈ –45% at LOEL)
Liver:
• Abs. liver weight ↑ (≈ +60% at LOEL)
• Rel. liver weight ↑ (≈ +15% at LOEL)
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +48% at LOEL)
Kidney:
• Abs. kidney weight (≈ +11% at 3 mg/kg bw/d, ≈ –12% at 30 mg/kg
bw/d)
• Rel. kidney weight ↑ (+9% at LOEL)
Haematopoietic system:
• Haemolysis in vena cava
• Haemolysis in orbital sinus

34

Control: 0.012 ± 0.006/ 0.032 (histopath
± 0.023/ 0.010 ± 0.004
ology)
1 mg/kg: 14.1 ± 4.3/17.2 ± 7.3/(Rosen et
17.7 ± 4.4
al., 2008)
3 mg/kg: 33.3 ± 15.0/ - / 47.9 ±(Gene
18.9
expression)
10 mg/kg: 99.0 ± 33.5/ 112.7 ±
20.4/ 85.6 ± 31.1
Liver concentrations of PFOA
in WT/CD-1/KO mice (µg/ml):

(complement/coagulation cascades), and xenobiotic metabolism
Doses: 0, 1, 3 mg/kg bw/d

Serum concentrations of PFOA (Wolf et
in WT/CD-1/KO mice (µg/ml): al., 2008)

10
10
3

30
30
10

1
0.3
0.3

3
1
1

-

-

1

3

1
0

3
0.3

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
thyroid,
0 mg/kg: 0.37 ± 0.14
endocrine system 0.3 mg/kg: 19 ± 2.5
1 mg/kg: 51 ± 10
3 mg/kg: 106 ± 10
10 mg/kg: 183 ± 46
30 mg/kg: 208 ± 51

(Loveless
et al.,
2006)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)

PFOA

14-day-study

(ammonium Rat (Crl:CD®(SD)IGS BR)
salt, linear
isoforms)
n/sex/group = 10 (m only)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

PFOA

14-day-study

(ammonium Rat (Crl:CD®(SD)IGS BR)
salt, branched
isoforms)
n/sex/group=10 (m only)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

Clinical chemistry:
• Serum cholesterol ↓ (≈ –55% at LOEL)
• Serum HDL ↓ (≈ –48% at LOEL)
• Serum non-HDL ↓ (≈ –43% at LOEL)
• Serum triglycerides ↓ (≈ –34% at LOEL)
Body weight (day 7) ↓ (≈ –31% at LOEL)
Body weight (day 13) ↓ (≈ –19% at LOEL)
Body weight gain (0-13) ↓ (≈ –26% at LOEL)
Liver:
• Abs. liver weight ↑ (≈ +28% at LOEL)
• Rel. liver weight ↑ (≈ +14% at LOEL)
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +90% at LOEL)
Kidney:
• Abs. kidney weight ↓ (≈ –16% at LOEL)
Haematopoietic system:
• Haemolysis in vena cava
• Haemolysis in orbital sinus
Clinical chemistry:
• Serum cholesterol ↓ (≈ –27% at LOEL)
• Serum HDL ↓ (≈ –42% at LOEL)
• Serum non-HDL (≈ –32% at LOEL)
• Serum triglycerides (≈ –21% at LOEL)

1
1
0.3
0.3
10
10
1

3
3
1
1
30
30
3

1
0.3
0.3

3
1
1

10

30

1
0.3

3
1

0
1
0
0

0.3
3
0.3
0.3

Remark: Conclusion of the study: Linear PFOA is similarly toxic as
linear/branched-mix (80:20), branched isoforms have weaker toxicity
Body weight gain (0-13) ↓ (≈ –24% at LOEL)
10
Liver:
• Abs. liver weight ↑ (≈ +19% at LOEL)
0.3
• Rel. liver weight ↑ (≈ +16% at LOEL)
0.3
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +34% at LOEL)
1
Kidney:
• Rel. kidney weight ↑ (≈ +10% at LOEL)
3
Haematopoietic system:
• Haemolysis in vena cava
3
• Haemolysis in orbital sinus
1
Clinical chemistry:
• Serum cholesterol ↓ (≈ –22% at LOEL)
0.3
• Serum HDL ↓ (≈ –30% at LOEL)
1
• Serum non-HDL (≈ –50% at LOEL)
1
• Serum triglycerides (≈ –44% at LOEL)
3

35

30
1
1
3
10
10
3
1
3
3
10

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
haematopoietic 0 mg/kg: 0.39 ± 0.51 µg/ml
system, thyroid, 0.3 mg/kg: 20 ± 3.2
endocrine system 1 mg/kg: 65 ± 11

(Loveless
et al.,
2006)

3 mg/kg: 137 ± 18
10 mg/kg: 206 ± 65
30 mg/kg: 223 ± 77

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
haematopoietic 0 mg/kg: 0.11 ± 0.24
system, thyroid, 0.3 mg/kg: 16 ± 2.5
endocrine system 1 mg/kg: 48 ± 12
3 mg/kg: 73 ± 25
10 mg/kg: 92 ± 20
30 mg/kg: 124 ± 33

(Loveless
et al.,
2006)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFOA

14-day-study

10
3
3

30
10
10

1
1
0

3
3
0.3

10

30

1
1
0
10
10
10

3
3
0.3
30
30
30

0.3
0
0

1
0.3
0.3

3
3

10
10

1
1
1
1

3
3
3
3

1
0
0.3

3
0.3
1

1
1
-

3
3
-

Remarks: Body weight changes only significant at 3 and 10 mg/kg, at
highest dose of 30 mg/kg no significant changes.
PFOA
28-day-study, USEPA, OPPTS 870.7800: Body weight ↓ (≈ –11% at LOEL after 28 days)
1
(ammonium Immunotoxicity, Health Effects Test
Body weight gain over 28 days ↓ (≈ –26% at LOEL)
1

10
10

(ammonium Mouse (Crl:DC®-1(ICR)BR)
salt,
linear/branch
n/sex/group = 10 (m only)
ed (80:20)
isoforms)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

PFOA

14-day-study

(ammonium Mouse (Crl:DC®-1(ICR)BR)
salt, linear
isoforms)
n/sex/group = 10 (m only)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

PFOA

14-day-study

(ammonium Mouse (Crl:DC®-1(ICR)BR)
salt, branched
isoforms)
n/sex/group = 10 (m only)
Exposure: oral (gavage, in water)
Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg
bw/d

Body weight (day 7) ↓ (≈ –16% at LOEL)
Body weight (day 13) ↓ (≈ –12% at LOEL)
Body weight gain (0-13) ↓ (≈ –200% at LOEL)
Liver:
• Abs. liver weight ↑ (≈ +112% at LOEL)
• Rel. liver weight ↑ (≈ +109% at LOEL)
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +92% at LOEL)
Kidney:
• Abs. kidney weight ↓ (≈ –20% at LOEL)
Clinical chemistry:
• Serum cholesterol ↓ (≈ –33% at LOEL)
• Serum HDL ↓ (≈ –43% at LOEL)
• Serum triglycerides ↑ (≈ +37% at LOEL)
Body weight (day 7) ↓ (≈ –16%)
Body weight (day 13) ↓ (≈ –18%)
Body weight gain (0-13) ↓ (≈ –500%)
Liver:
• Abs. liver weight ↑ (≈ +61% at LOEL)
• Rel. liver weight ↑ (≈ +17% at LOEL)
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +143% at LOEL)
Kidney:
• Abs. kidney weight ↓ (≈ –18% at LOEL)
• Rel. kidney weight ↓ (≈ –11% at LOEL)
Clinical chemistry:
• Serum cholesterol ↓ (≈ –35% at LOEL)
• Serum HDL ↓ (≈ –46% at LOEL)
Body weight (day 13) ↑ (≈ +6%)
Body weight gain (0-13) ↑ (≈ +200%)
Liver:
• Abs. liver weight ↑ (≈ +149% at LOEL)
• Rel. liver weight ↑ (≈ +19% at LOEL)
• Hepatic peroxisomal ß-oxidation activity ↑ (≈ +103% at LOEL)
Clinical chemistry:
• Serum cholesterol ↓ (≈ –34% at LOEL)
• Serum HDL ↓ (≈ –34% at LOEL)
• Serum non-HDL (≈ –35% only at 3 mg/kg)

36

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
haematopoietic 0 mg/kg: 0.04 ± 0.02
system, thyroid, 0.3 mg/kg: 10 ± 1.4
endocrine system 1 mg/kg: 27 ± 5

(Loveless
et al.,
2006)

3 mg/kg: 66 ± 8.6
10 mg/kg: 190 ± 29
30 mg/kg: 241 ± 28

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
haematopoietic 0 mg/kg: 0.07 ± 0.06
system, thyroid, 0.3 mg/kg: 13 ± 2.4
endocrine system 1 mg/kg: 32 ± 5.2

(Loveless
et al.,
2006)

3 mg/kg: 69 ± 10
10 mg/kg: 225 ± 68
30 mg/kg: 259 ± 34

• nervous system,

Serum PFOA concentrations
immune system, (µg/ml):
haematopoietic 0 mg/kg: 0.02 ± 0.03
system, thyroid, 0.3 mg/kg: 14 ± 3.5
endocrine system 1 mg/kg: 34 ± 10

(Loveless
et al.,
2006)

3 mg/kg: 82 ± 10
10 mg/kg: 172 ± 29
30 mg/kg: 244 ± 50

• Kidney, nervous

system,

(Loveless
et al.,

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
salt, linear)
19.5%
aqueous
solution (in
NANOpure
water)

Guidelines (1998)

Liver:
• Abs. liver weight ↑ (≈ +30%% at LOEL)
Rat (Crl:CD(SD)IGS BR)
• Rel. liver weight ↑ (≈ +83% at LOEL)
Immune system:
n/sex/group = 10 (m only)
• Rel. thymus weight ↑ (≈ +44% at LOEL)
• Abs./rel. spleen weight (ns)
• Anti-sRBC IgM antibody (ns)
Exposure: oral (gavage)
Endocrine system:
Serum corticosterone (ns)
Doses: 0, 0.3, 1, 10, 30, 30/0 mg/kg
Clinical chemistry:
bw/d
• Cholesterol ↓ (≈ –36/–31% at 0.3/1 mg/kg bw/d)
• HDL ↓ (≈ –25% at LOEL)
• Non-HDL ↓ (≈ –42%/–37% at 0.3/1 mg/kg bw/d)
• Triglycerides ↓ (≈ –31% at LOEL)
PFOA
28-day-study, USEPA, OPPTS 870.7800: Body weight ↓ (≈ –14% at LOEL after 28 days)
(ammonium Immunotoxicity, Health Effects Test
Liver:
salt, linear) Guidelines (1998)
• Abs.+ rel. liver weight ↑ (≈ +80% at LOEL)
19.5%
Immune system:
aqueous
• Abs. spleen weight ↓ (≈ –44% at LOEL)
Mouse (Crl:CD-1(ICR)BR)
solution (in
• Rel. spleen weight ↓ (≈ –14% at LOEL)
NANOpure
• Abs. + rel. thymus weight ↓ (≈ –45% at LOEL)
water)
• Anti-sRBC IgM antibody ↓ (≈ –22% at LOEL)
n/sex/group=20 (m only)
Endocrine system:
Serum corticosterone ↑ (≈ +120% at 10 mg/k/d)
Exposure: oral (gavage)
Clinical chemistry:
• Cholesterol ↓ (≈ –31% at LOEL)
• HDL ↓ (≈ –29% at LOEL)
Doses: 0, 0.3, 1, 10, 30, 30/0 mg/kg
• Non-HDL (ns)
bw/d
• Triglycerides ↓ (≈ –53% at LOEL)
Liver:
PFOA (90%) 3-week study, non-guideline
• Abs. liver weight ↑ (≈ +77% at LOEL)
Mouse (no strain given)
• Total glutathione ↑ (≈ +101% at LOEL)
• Hepatic catalase ↑ (≈ +482% at LOEL)
n/sex/group = 5 (f only)
• Hepatic SOD (ns)
exposure: oral (gavage, PFOA in water) • Hepatic Glutathione reductase ↑ (≈ +156% at LOEL)

0.3
1

1
10

10

30 (30/0)

-

0.3

1

0.3
10

0.3

1

1
0.3
1
1

10
1
10
10

1
0.3

10
1

1

10

1
1
1
10
5

5
5
5

n/sex/group = 15 (m only)

Body weight ↓ (≈ –9% at LOEL)
Body weight gain ↓ (≈ –68% at LOEL)
Liver:
• Abs. liver weight ↑ (≈ +70% at LOEL)

1
1

10
10

1

10

37

• Kidney, nervous

system,
haematopoietic
system, thyroid

• Body weight,

10

doses: 0, 1, 5, 10 mg/kg bw/d
PFOA (99.9%) 14-days, rat (CD)

haematopoietic
system, thyroid

kidney, nervous
system, immune
system,
haematopoietic
system, thyroid,
endocrine system
• kidney, nervous

system, immune
system,
haematopoietic

2008)

(Loveless
et al.,
2008)

(Ahmed
and Abd
Ellah,
2012)

(Cook et
al., 1992)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Rel. liver weight ↑ (≈ +181% at LOEL)

Exposure: oral (gavage)

1
1

• Hepatic β-oxidation activity ↑ (≈ +246% at LOEL)

Endocrine system:
• Serum estradiol ↑ (≈ +64% at LOEL)
1
Doses: 0, 1, 10, 25, 50 mg/kg bw/d
Reproductive tissue:
• Abs./rel. testes weight ↑ (≈ +20%/+21% at LOEL)
10
• Abs. accessory sex organ weight ↓ (≈ –16% at LOEL)
10
• Abs. ventral prostate weight ↓ (≈ –12% at LOEL)
25
Body weight ↓ (≈ –16%)
PFOA (99.9%) 14-days
Liver:
Rat (Crl:CD BR(CD))
• Rel. liver weight ↑ (≈ +79%)
• Hepatic β-oxidation ↑ (≈ +760%)
n/sex/group = 15 (m only)
• Hepatic aromatase ↑ (≈ +354%)
Endocrine system:
• Serum estradiol ↑ (≈ +184%)
Exposure: oral (gavage)
Reproductive tissue:
• Testicular interstitial fluid testosterone ↑ (≈ +93%)
Doses: 0, 25 mg/kg bw/d
• Testicular interstitial fluid estradiol ↑ (≈ +115%)
PFOA (98Body weight gain (ns)
24-months-study
100%)
Liver:
Rat (Crl:CD BR (CD))
• Liver adenoma ↑ (≈ +1200% (13/1))
Endocrine system:
n/sex/group = 156 (m only)
• Estradiol ↑ (≈ +271/+110/+70/+71/+78% after 1/3/6/9/12 month(s)
• Prolactin ↓ (≈ –40/–29% after 3/6 months)
n/group (hormonal analysis) = 10
Reproductive tissue:
n/group (peroxisome prolif.) = 6
• Testis weight ↑ (≈ +8% after 24 months)
• Leydig cell adenoma ↑ (≈ +267%)
Exposure: oral (dietary)
Other effects:
• Pancreatic cell proliferation ↑ (≈ +175/+125% after 15/18 months)
• Pancreatic acinar cell hyperplasia ↑ (≈ +290%)
Doses: 0, 300 ppm (equivalent to 13.6
• Pancreatic acinar cell adenoma ↑ (≈ +800%)
mg/kg bw/d)
PFOA (96%)

28-day RDT-study, non-guideline
Rat (SD)
n/sex/group = 10 (m only)
Exposure: oral (gavage, in water)

Remark: Mechanistic study on tumor induction
Body weight ↓ (≈ –6% at LOEL after 27 days)
Liver:
• Rel. liver weight ↑ (≈ +75% at LOEL)
• Cytoplasmic vacuolation
• Focal or flakelike necrosis
• Hepatocellular hypertrophy
Kidney:

5
–
–
–
–

10
10

system, thyroid,

10
25
25
50
• kidney, nervous

(Biegel et
al., 1995)

system, immune
system,
haematopoietic
system, thyroid,

(Biegel et
al., 2001)

•

20
5
5
5
5

• , nervous system, Blood PFOA concentrations

immune system, (µg/ml):
haematopoietic 0 mg/kg: 0
system, thyroid 5 mg/kg: 39.2 ± 14.4
20 mg/kg: 58.8 ± 17.6
Liver PFOA concentrations

38

(Cui et al.,
2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Rel. kidney weight ↑ (≈ +15% at LOEL)

Doses: 0, 5, 20 mg/kg bw/d

Reproductive tissue:
• Rel. testis weight ↑ (≈ +20% at LOEL)
Other effects:
• Pulmonary congestion
• Focal or diffuse thickened epithelial walls of respiratory system

–

5

(µg/g):

–

5

–
–

5
5

0 mg/kg: 0
5 mg/kg: 218 ± 21
20 mg/kg: 196 ± 10
Kidney PFOA concentrations
(µg/g):
0 mg/kg: 0
5 mg/kg: 228 ± 37
20 mg/kg: 209 ± 74
Lung PFOA concentrations
(µg/g):
0 mg/kg: 0
5 mg/kg: 63.0 ± 11.3
20 mg/kg: 64.3 ± 15.9
Heart PFOA concentrations
(µg/g):
0 mg/kg: 0
5 mg/kg: 35.5 ± 17.6
20 mg/kg: 34.6 ± 18.0
Spleen PFOA concentrations
(µg/g):
0 mg/kg: 0
5 mg/kg: 13.6 ± 2.4
20 mg/kg: 6.92 ± 9.31
Testicle PFOA concentrations
(µg/g):
0 mg/kg: 0
5 mg/kg: 16.7 ± 16.9
20 mg/kg: 16.8 ± 19.2
Brain PFOA concentrations
(µg/g):
0 mg/kg: 0

39

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
5 mg/kg: 10.5 ± 9.8
20 mg/kg: 7.20 ± 6.03
PFOA (97%)

7 days
Rat (SV129 WT and PPARα-null)
n/sex/group = 4 (m only)
Exposure: oral (gavage)

Body weight (ns)
Liver:
• Abs. liver weight (WT/PPARAα-null) ↑ (≈ +170%/+175%)
• Rel. liver weight (WT/PPARAα-null) ↑ (≈ +143%/+171%)
• Cell area (WT/PPARAα-null) ↑ (≈ +105%/+96%)
• Hepatic DNA content (WT/PPARAα-null) ↓ (≈ –43%/–35%)
• Hepatic lipid accumulation (WT) ↑ (≈ +900%)
• Hepatic triglycerides (WT) ↑ (≈ +225%)

• kidney, nervous

(Das et al.,
2017)

system, immune
system,
haematopoietic
system, thyroid

Doses: 0, 10 mg/kg bw/d
PFOA (≥98%) 15 days, non-guideline study
Mouse (C57BL/6N)
n/sex/group = 8 (f only)

Body weight ↓ (1 day postdosing) (≈ –4% at LOEL)
Immune system:
• Abs. + rel. spleen weight ↓ (≈ –16% at LOEL at 1 day PD)
• Abs. + rel. thymus weight ↓ (≈ –30% at LOEL at 1 day PD)
• SRBC IgM antibody titers ↓ (≈ –10% at LOEL)
• SRBC IgG antibody titers ↑ (≈ +15% at LOEL)

7.5

15

1.88
7.5
1.88
1.88

3.75
15
3.75
3.75

Exposure: oral (drinking water)

• Liver, kidney,

Serum PFOA concentrations (DeWitt et
nervous system, (ng/mL) at 1 day postdosing: al., 2008)
haematopoietic 0 mg/kg: 54.3 ± 4.9
system, thyroid 3.75 mg/kg: 74.9 ± 2.7
7.5 mg/kg: 87.2 ± 3.3
15 mg/kg: 128.1 ± 6.8
30 mg/kg: 162.6 ± 8.4
Serum PFOA concentrations
(ng/mL) at 15 day postdosing:

Doses: 0, 0.94, 1.88, 3.75, 7.5, 15, 30
mg/kg bw/d

PFOA (≥98%) 10-day-study
Mouse (C57BL/6N; adx/shamadrenolectomized)
n/sex/group = 5-7 (f only)
Exposure: oral (drinking water)
Doses: 0, 3.75, 7.5, 15 mg/kg bw/d

0 mg/kg: 156.4 ± 14.9
3.75 mg/kg: 35.9 ± 1.6
7.5 mg/kg: 42.8 ± 1.7
15 mg/kg: 50.0 ± 1.5
30 mg/kg: 52.7 ± 3.2
Body weight (sham) ↓ (≈ –9.8% at LOEL at 2 days postdosing)
Body weight (adx) ↓ (≈ –10.3% at LOEL, at 2 days postdosing)
Immune system:
• SRBC IgM titer (sham) ↓ (≈ –18% at LOEL)
• SRBC IgM titer (adx) ↓ (≈ –14% at LOEL)
Endocrine system:
• Serum corticosterone ↑ (≈ +168% at LOEL)
Clinical Chemistry (after 5 days of dosing, no effects after 10 days)
• ALT (adx) ↑ (≈ +250% at LOEL)
• AST (adx) ↑ (≈ +72% at LOEL)
• SDH (adx) ↑ (≈ +135% at LOEL)
• Cholesterol (sham) ↓ (≈ –19% at LOEL)
• Triglycerides (sham) (≈ –45% at LOEL)

40

3.75
7.5

7.5
15

7.5
3.75

15
7.5

7.5

15

7.5
7.5
7.5
7.5
–

15
15
15
15
3.75

• Liver, kidney,

nervous system,
haematopoietic
system, thyroid

(DeWitt et
al., 2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Triglycerides (adx) (≈ –60% at LOEL)

3.75
• LDH (adx) ↑ (≈ +36% at LOEL)
7.5
PFOA
15-day-study (immunization on day 11) Body weight ↓ (≈ –14% at LOEL from day 12 onwards, only in WT) 7.5
(ammonium Mouse (WT (C57BL/6-Tac)) and PPARα Immune system:
salt, ≥98%) KO (B6.129S4-Ppartm1GonzN12))
• Rel. spleen weight (only WT) ↓ (≈ –32% at LOEL)
7.5
• Rel. thymus weight (only WT) ↓ (≈ –58% at LD, no effect at HD)
• T-cell dependent IgM antibody response (WT) ↓ (≈ –17%)
7.5
n/sex/group = 6 (f only)
• T-cell dependent IgM antibody response (PPARα-KO) ↓ (≈ –14%)
7.5
Exposure: oral (drinking water)

7.5
15
30
30
30
30

• Liver, kidney,

nervous system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(DeWitt et
al., 2016)

Remark: Suppression of T-cell dependent antibody response seemed
to be PPARα-independent. “…effects on humoral immunity are likely
mediated by disruption of B-cell/plasma cell function.”

Doses: 0, 7.5, 30 mg/kg bw/d
PFOA
15-day-study (immunization on day
(ammonium 11), mouse (WT (C57BL/6N))
salt, ≥98%) n/sex/group = 8 (f only)
Exposure: oral (drinking water)
Doses: 0, 0.94, 1.88, 3.75, 7.5 mg/kg
bw/d
PFOA
15-day-study (immunization on days
(ammonium 11)
salt, ≥98%) Mouse (WT (C57BL/6N))
n/sex/group = 4 (f only)
Exposure: oral (drinking water)

Body weight (ns)
Immune system:
• Rel. spleen weight ↓ (≈ –17% at LOEL)
• Rel. thymus weight ↓ (≈ –14% at LOEL)
• T-cell-independent IgM antibody response ↓ (≈ –10% at LOEL)

• Liver, kidney,

3.75
3.75
0.94

Immune system: (Splenic lymphocyte phenotypes)
• CD4-/CD8+ ↑ (≈ +14% at HD after 13 days (i.e. 2 days after
immunization)) ↓ (≈ –10% only at LD after 15 days (i.e 5 days after
immunization))
• CD4-/CD8- ↓ (≈ –30% at LOEL after 13 days, but no effect after 15 days)
• CD4+/CD8- ↑ (≈ +7% only at LD after 15 days)
• NK cells ↓ (≈ –21% only at LD after 15 days)

7.5
7.5
1.88

3.75

nervous system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry
• body weight,
liver, kidney,
nervous system,
haematopoietic
system, thyroid,
clinical chemistry

(DeWitt et
al., 2016)

(DeWitt et
al., 2016)

•

Remark: there were no PFOA effects before immunization on day 10
Doses: 0, 3.75, 7.5 mg/kg bw/d
PFOA

7-day study
Rat (Wistar)
n/sex/group = 4 (m only)
Exposure: oral (dietary)

Body weight ↓ (≈ –6%)
Liver:
• Abs. liver weight ↑ (≈ +66%)
• Liver non-esterified cholesterol ↑ (≈ +48%)
• Cholesterol synthesis in hepatocytes ↓ (≈ –75% from acetate/-55%
from pyruvate/-80% from hydroxyl-methylglutarate)
• Fatty acid synthesis from acetate in hepatocytes ↓ (≈ –64%)
• Enzymatic activity in hepatocytes (≈ –60% for acetate thiokinase,
≈ +272% for malate dehydrogenase(NADP)(decarboxylating), ≈ –

41

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Haughom
and
Spydevold,
1992)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 0.02% (equivalent to 17
mg/kg bw/d)

PFOA
28-day-studies (study 1 and 2)
(ammonium Rat (Sprague-Dawley)
salt, 98%)
n/sex/group = 10 (m only)
Exposure: oral (dietary)
Doses: 0, 300 ppm (equivalent to 20
mg/kg bw/d)

PFOA
7-day RDT study
(ammonium Mouse (C57BL/6)
salt)
n/sex/group = 6 (m only)
Exposure: oral (gavage)

54% for Glucose-6-phosphate dehydrogenase)
• Acetyl-CoA Cholesterol acyltransferase (ACAT) activity) ↓ (≈ –51%)
Clinical chemistry:
• Serum cholesterol ↓ (≈ –56%)
• Serum triglycerols ↓ (≈ –39%)
Remark: Also PFOSA data available
Body weight ↓ (≈ –12%/–11% on day 8; ≈ –23%/–13% on day 29)
Liver:
• Abs. liver weight ↑ (≈ +25%/+37% on day 8 in study 1/2; ≈ +55% on
day 29 but only in study 2, not in study 1)
• Rel. liver weight ↑ (≈ +42%/+53% on day 8; ≈ +47%/+66% on day
29)
• Hepatic cell proliferation ↑ (≈ +236%/+114% on day 2; ≈
+318%/+413% on day 8; ≈ +164% on day 29 (only study 2 data
available)
• Relative liver DNA concentration ↓ (≈ –13% on day 2 (only study 1
significant effect); ≈ –22%/-26% on day 8; ≈ –13%/–32% on day 29)
• Peroxisome proliferation (PCO) ↑ (≈ +120%/+166% on day 2;
≈ +558%/+677% on day 8; ≈ +888%/+656% on day 29)
Clinical chemistry (study 1 only):
• Plasma AST ↓ (≈ –25%/-19% on day 2/29)
• Plasma cholesterol ↓ (≈ –61%/–40% on day 8/29)
• Plasma glucose ↓ (≈ –38%/–38% on day 8/29)
• Plasma triglycerides ↑/↓ (≈ +37% on day 2; ≈ –75%/–73% on day
8/29)
Liver:
• Abs. + rel. liver weight ↑ (≈ +35% at LOEL)
• Hepatic mRNA/protein expression of Cyp4a14 ↑
(≈ +11900%/+1150% at LOEL)
• Hepatic mRNA/protein expression of Bcrp ↑ (≈ +90%/+70% at LOEL) Kidney:
• Renal mRNA/protein expression of Cyp4a14 ↑ (≈ +90%/ns at LOEL) 1

• kidney, nervous

Plasma levels of APFO
(Elcombe
system, immune (µg/mL):
et al.,
system,
Control: < quantification limit 2010)
haematopoietic
Day2: 259 ± 39
system, thyroid,
endocrine system Day 8: 234 ± 33
Day 29: 252 ± 45

• Body weight,

1
1
1

nervous system,
immune system,
haematopoietic
system, thyroid,
endocrine system

(Eldasher
et al.,
2013)

3

Doses: 0, 1, 3 mg/kg bw/d
PFOA
10-day RDT study (hypersensitivity
(ammonium study)
salt, >96%) Mouse (BALB/c)

Liver:
• Abs. liver weight ↑ (≈ +53% at LOEL)
Immune system:
• Thymus weight ↓ (≈ –41% at LOEL)

• Body weight,

42

2.5

6.25

25

50

kidney, nervous
system,
haematopoietic

(Fairley et
al., 2007)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
n/sex/group= 5 (f only)
Exposure: dermal (PFOA in acetone,
exposure on ear), 4 days exposure,
sacrifice 6 days after final exposure

• Spleen weight ↓ (≈ –28% at LOEL)

12.5

25

system, thyroid,
endocrine system

Remark: Hypersensitivity study results not listed here

Doses: 0, 0.25, 2.5, 6.25, 12.5, 25, 50
mg/kg bw/d
PFOA

14-day RDT study
Rat (SD)

Liver:
• Rel. liver weight ↑ (≈ +45%)
• CAT activity ↑ (≈ +57%)
• Induction of 80K-protein

• Body weight,

kidney, nervous
system, immune
system,
haematopoietic
system, thyroid,
endocrine system

n/sex/group = 3 (m only)
Exposure: oral (dietary)

Remark: Acute toxicity dose response for PFOA also available (i.p.
injection, 5-150 mg/kg)

(Ikeda et
al., 1985)

Doses: 0, 0.02% (equivalent to 20
mg/kg bw/d)
PFOA
14-day RDT study
(ammonium Rat (Crj:CD(SD)IGS)
salt, 10%
aqueous
n/sex/group = 4 (m only)
solution)

Body weight (increase but ns)
Liver:
• Rel. liver weight ↑ (≈ +102% at LOEL)
5
• Cyanide-insensitive palmitoyl CoA β-oxidation activity ↑ (≈ +598% 5
at LOEL)
• Carnitine acetyltransferase activity ↑ (≈ +1960% at LOEL)
5

• kidney, nervous

50
50
50

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Iwai and
Yamashita,
2006)

Exposure: oral (gavage)
Doses: 0, 0.5, 5, 50 mg/kg bw/d
PFOA

1-week RDT-study
Rat (Wistar)
n/sex/group= (males only)
Exposure: oral (dietary)
Doses: 0, 0.0025, 0.005, 0.01, 0.02,

Body weight (ns)
Liver:
• Abs. + rel. liver weight ↑ (≈ +33% at LOEL)
• Peroxisomal β-oxidation ↑ (≈ +300% at LOEL)
• Hepatic acyltransferase ↑ (≈ +30% at LOEL)
• Hepatic hydrolase ↑ (≈ +700% at LOEL)
• Hepatic GSH S-transferase ↓ (≈ –22% at LOEL)
• Hepatic triacylglycerol ↑ (≈ +160% at LOEL)
• Hepatic cholesterol ↑ (≈ +50% at LOEL)
• Hepatic phospholipid ↑ (≈ +33% at LOEL)

• kidney, nervous

2.5
2.5
–
2.5
–
–
5
5

43

5
5
2.5
5
2.5
2.5
10
10

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Kawashim
a et al.,
1995)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
0.04 % (equivalent to 2.5, 5, 10, 21,
and 43 mg/kg bw/d)
PFOA
21-day RDT studies
(ammonium Mouse (Crl:CD-1)
salt, 99%)
n/sex/group=5

Mortality ↑ (100% mortality at 3000 ppm within 14 days)
Body weight ↓ (data not shown)
Liver:
• Abs. liver weight ↑ (m/f, ≈ +35/+32% at LOEL)
• Rel. liver weight ↑ (m/f, ≈ +31/+29% at LOEL)

38
3.8

380
38

0.13/0.13 0.38/0.38
0.13/0.13 0.38/0.38

• kidney, nervous

(Kennedy,
1987)

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

Exposure: oral (dietary)
Doses: 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10,
30, 300, 3000 ppm (300 and 3000 ppm
only in 14 day study)
(equivalent to 0, 0.0013, 0.0038, 0.013,
0.038, 0.13, 0.38, 1.3, 3.8, 38, 380
mg/kg bw/d)
PFOA
(>99.9%)

Body weight ↓ (-14% at LOEL)
Liver:
• Abs. + rel. liver weight ↑ (≈ +34% at LOEL)
• Protein of hepatic microsomes ↑ (≈ +7% at LOEL)
n/sex/group=15 (males only)
• Hepatic aromatase activity ↑ (≈ +300% at LOEL)
• Total hepatic aromatase ↑ (≈ +560% at LOEL)
• Hepatic β-oxidation activity ↑ (≈ +190% at LOEL)
Exposure: oral (gavage)
• Total hepatic cytochrome P450 ↑ (≈ +70% at LOEL)
Endocrine system:
Doses: 0, 0, 0.02, 2, 20, 40 mg/kg bw/d Serum estradiol ↑ (≈ +56% at LOEL)
(controls ad libitum and pair-fed to 40 Reproductive tissue:
mg control)
• Rel. testes weight ↑ (≈ +12% at LOEL) (compared to ad libitum
control); ↓ (≈ –11% at 40 mg compared to pair-fed control)
PFOA
4-week RDT study
Body weight (WT) ↓ (≈ –14% at LOEL)
(ammonium Mouse (Wild-type mice (129S4/SvlmJ) Body weight gain (WT) ↓ (≈ -167% at LOEL)
salt, >98%) and Pparα-null mice) (129S4/SvJaeBody weight (PPARα KO) ↑ (≈ +11% at LOEL, ns at highest dose)
Liver:
Pparαtm1Gonz/J)
• Abs. + rel. liver weight (WT) ↑ (≈ +240% at LOEL)
• Abs./rel. liver weight (PPARα KO) ↑ (≈ +180% at LOEL)
n/sex/group = 10 (males only)
Clinical Chemistry:
• Plasma AST (WT) ↑ (≈ +83% at LOEL)
Exposure: oral (gavage)
• Plasma ALT (WT) ↑ (≈ +577% at LOEL)
• Plasma total bilirubin (WT)( ≈ –44% at 12.5 µmol/kg; ≈ +67% at 50
14-day RDT study

Rat (Crl:CD BR(CD))

44

2

20

0.2
0.2
0.2
0.2
2

2
0.2
2
2
2
20

0.2

2

2

20

10.8
5.4
–

21.6
10.8
5.4

–
–

5.4
5.4

5.4
–

10.8
5.4

• kidney, nervous

(Liu et al.,
1996)

system, immune
system,
haematopoietic
system, thyroid

• kidney, nervous

PFOA concentrations in whole (Minata et
system, immune blood (WT) (µg/ml):
al., 2010)
system,
12.5 µmol/kg: 20.6 ± 2.4
haematopoietic 25 µmol/kg: 46.9 ± 3.2
system, thyroid, 50µmol/kg:64.2 ± 6.5
endocrine system
PFOA concentration in bile
(WT) (µg/ml):
12.5 µmol/kg: 56.8 ± 26.9
25 µmol/kg: 784.0 ± 137.6

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 12.5, 25, 50 µmol/kg/d (0,
5.4, 10.8, 21.6 mg/kg bw/d)

µmol/kg)
• Plasma total cholesterol (WT) ↓ (≈ –17% at LOEL)
• Plasma triglycerides (WT) ↑ (≈ +47% at LOEL, ns at highest dose)
• Plasma AST (PPARα KO) ↑ (≈ +535% at LOEL)
• Plasma ALT (PPARα KO) ↑ (≈ +491% at LOEL)
• Plasma total bilirubin (PPARα KO)( ≈ +683% at 50 µmol/kg)
• Plasma total bile acid (PPARα KO) (≈ +1350% at 50 µmol/kg)
• Plasma total cholesterol (PPARα KO) (≈ –38%/-36% at 12.5/25
µmol/kg; ≈ +66% at 50 µmol/kg)
• Plasma triglycerides (PPARα KO) ↑ (≈ +102% at LOEL)

50µmol/kg: 2174.0 ± 332.4
5.4
–
10.8
–

10.8
5.4
21.6
5.4

PFOA concentration in liver
(WT) (µg/ml):
12.5 µmol/kg: 181.2 ± 6.3
25 µmol/kg: 198.8 ± 15.4
50µmol/kg: 211.6 ± 13.3
PFOA concentrations in whole
blood (Pparα(-/-)) (µg/ml):

–

5.4

12.5 µmol/kg: 19.3 ± 2.2
25 µmol/kg: 36.4 ± 2.7
50µmol/kg: 71.2 ± 8.0
PFOA concentrations in bile
(Pparα(-/-)) (µg/ml):
12.5 µmol/kg: 19.6 ± 2.2
25 µmol/kg: 62.9 ± 16.7
50µmol/kg: 383.0 ± 109.9
PFOA concentrations in liver
(Pparα(-/-)) (µg/ml):
12.5 µmol/kg: 172.3 ± 8.9
25 µmol/kg: 218.3 ± 14.5
50µmol/kg: 239.7 ± 25.0

PFOA
7-day RDT study
(ammonium Rat (Crl:CD(SD)BR)
salt, 70:30
linear:branch
n/sex/group = 6 (males only)
ed)
Exposure: oral (gavage)
Dose: 0 (pair-fed), 50 mg/kg bw/d

Body weight (no difference compared to control; decrease in body
weight in both control and treatment over 7 days)
Liver:
• Abs./rel. liver weight ↑ (≈ +119/+91% after 7 days)
• Hepatic DNA ↓ (≈ –57% after 7 days)
• Hepatic cytochrome P-450 activity ↑ (≈ +120% after 1 day, +220%
after 3 days)
• Hepatic benzphetamine N-demethylase activity ↑ (≈ +100% after 3
days)
• Hepatic ethyresorufin O-deethylase activity ↓ (≈ –47% after 1 day; 51% after 3 days)
• Hepatic carnitine acetyltransferase activity ↑ (≈ +1117% after 3
days)
• Hepatic carnitine palmitoyltransferase activity ↑ (≈ +94% after 3
days)
• Incorporation of [14C] acetate into hepatic neutral lipids ↑
(≈ +2763% for triacylglycerols, ≈ +1200% for cholesterylesters, all

45

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Pastoor et
al., 1987)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
after 7 days)
• Incorporation of [14C] acetate into hepatic neutral lipids ↑ (≈ +764%
for phosphatidyl ethanolamine, ≈ +2433% for phosphatidyl serine,
≈ +1514% for phosphatidyl inositol, ≈ +937% for phosphatidyl
choline, ≈ +543% for sphingomyelin, all after 7 days)
Clinical chemistry
• Plasma cholesterol and triacylglycerol (both ns after 7 days)
Remark: The increased 14C incorporation is interpreted as increased
lipogenesis, which contrasts hypolipidemic effects of other
peroxisome proliferators, e.g. decreased lipogenesis with DHEP
PFOA
13-week-RDT study,+8-week recovery Body weight ↓ after 1-13 weeks, but not in recovery (≈ –9% after 13 1.94
(ammonium Rat (Crl:CDBR)
weeks compared to ad libitum, ns compared to pair-fed control)
salt, 98%
Body weight gain ↓ over weeks 1-13 (≈ –14% compared to ad
1.94
purity)
libitum control, ns compared to pair-fed control)
n/sex/group = 5-15 (m only)
Liver:
• Rel. liver weight ↑ (≈ +13% at LOEL after 4 weeks, after 7 and 13
0.06
weeks, LOEL at 30 ppm!, after 13 weeks significant reduction of -8%
Exposure: oral (dietary)
at 1 ppm…)
• Hepatic palmitoyl CoA oxidase activity ↑ (≈ +75% at LOEL compared 0.06
Doses: 0, 1, 10, 30, 100 ppm (ad
to ad libitum control after 4 weeks, after 7 and 13 weeks LOEL at 30
libitum control and pair-fed control
ppm (≈ +75% compared to pair-fed control after 13 weeks))
with 100 ppm) equivalent to 0, 0.06, • Hepatocyte hypertrophy (minimal at LOEL, mild at 30 and 100 ppm) 0.06
0.64, 1.94, 6.5 mg/kg bw/d
Endocrine system:
• Estradiol, LH and testosterone (ns)
Remark: Effects are reversible in recovery (Serum levels of PFOA
decline rapidly in recovery). Overall, “study no effect level was 1 ppm
(0.06 µg/mg) with doses of 10 ppm (0.64 µg/mg) and higher
producing adaptive and reversible liver changes.”

6.5
6.5

0.64

0.64

0.64

• kidney, nervous

Serum PFOA concentrations
system, immune after 4 weeks:
system,
0 ppm: haematopoietic 0 (pair fed): system, thyroid, 1 ppm: 6.5 ± 1
clinical chemistry 10 ppm: 55.81 ± 8.1
30 ppm: 104 ± 14
100 ppm: 159+ ± 30
Serum PFOA concentrations
after 7 weeks:
0 ppm: 0 (pair fed): 1 ppm: 7.5 ± 1.3
10 ppm: 46 ± 16
30 ppm: 87 ± 28
100 ppm: 149 ± 35
Serum PFOA concentrations
after 13 weeks:
0 ppm: 0 (pair fed): 1 ppm: 7.1 ± 1.2
10 ppm: 41 ± 13
30 ppm: 70 ± 16
100 ppm: 138 ± 34
Serum PFOA concentrations
after 21 weeks (13 weeks RDT
+ 8 weeks recovery):
0 ppm: 0 (pair fed): not applicable

46

(Perkins et
al., 2004)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
1 ppm: 1.2
10 ppm: 1.1 ± 1.3
30 ppm: 1.6 ± 0.9
100 ppm: 2.5 ± 0.9
PFOA (98%
purity)

PFOA (98%
purity)

Body weight ↓ (≈ –6% at LOEL after 5 days)
–
Liver:
• Abs./rel. liver weight ↑ (≈ +75%/+112% at LOEL after 5 days)
–
• Hepatic protein content ↑ (≈ +36% at LOEL after 5 days)
–
n/sex/group = 3 (m only)
• Hepatic mitochondrial protein content ↑ (≈ +767% at LOEL after 5 –
days)
• Hepatic microsomal protein content ↑ (≈ +55% at LOEL after 5
24
Exposure: oral (dietary)
days)
• Hepatic peroxisomal catalase specific activity↑ (≈ +154% at LOEL
–
Doses: 0, 0.02, 0.1% (data from all
after 5 days)
treatments only for 5 days) (equivalent • Hepatic lauroyl-CoA oxidase specific activity ↑ (≈ +171% at LOEL –
to 0, 24, 120 mg/kg bw/d (calculations after 5 days)
by ATSDR 3x higher))
• Hepatic palmitoyl-CoA oxidation specific activity ↑ (+244% at LOEL –
after 5 days)
(0.001, 0.003, 0.01% only for hepatic
• Hepatic cyanide-insensitive palmitoyl-CoA oxidation specific activity –
enzymes)
↑ (≈ +312% at LOEL after 10 days)
2/5/10 days

Mouse (C57B1/6)

Body weight and Liver mass and liver enzymes see Permadi et al.
1993 (same study)
Liver:
• Microsomal cytochrome P450 ↑ (≈ +293% in Table5, +200% in Table
n/sex/group = 3 (m only)
6)
• Microsomal cytochrome P450 reductase ↑ (≈ +100% in Table5,
+285% in Table 6)
Exposure: oral (dietary)
• Microsomal Cytochrome B5 (↑) (ns in Table 5, +70% in Table 6)
• Microsomal epoxide hydrolase ↑ (≈ +239% in Table5, +191% in
Doses: 0, 0.02, (0.1)% (control only for Table 6)
10 days, 0.1% only 2 and 5 days)
• Hepatic cytosolic DT-diaphorase ↑ (≈ +391% in Table7, +438% in
Table 8)
(equivalent to 24, 120 mg/kg bw/d)
• Hepatic cytosolic glutathione peroxidase (↓) (ns in Table7, –29% in
Table 8)
• Hepatic cytosolic SOD (↑) (ns in Table7, ≈ +53% in Table 8)
• Hepatic cytosolic epoxide hydrolase ↑ (≈ +105% in Table7, +107% in
Table 8)
• Lipid peroxidation in mitochondrial fraction from liver (TBARS) ↓
(≈ –34%/–58% in Exp.1/Exp.2)
• Lipid peroxidation in mitochondrial fraction from liver (O2
10-day study

Mouse (C57B1/6)

47

24
24
24
24

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Permadi
et al.,
1993)

120
24
24
24
1.2
• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Permadi
et al.,
1992)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
consumption in Fe presence) ↓ (≈ –54%/–55% in Exp.1/Exp.2)

PFOA (96%
purity)

PFOA (96%
purity)

Remark: Also data for 2 days (0.1%) or 5 days (0.02% and 0.1%)
available, but in this publication only control levels for 10 days are
reported. It is not clear, why data in Table 5 and 6/ table 7 and 8 are
different…
Body weight ↓ (data not shown)
10-day study
Liver weight ↑ (data not shown)
Mouse (C57BL/6 (H-2b))
Immune system:
• Number of total white blood cells ↓ (≈ –72%)
0.34
n/sex/group = 4 (m only)
• Number of lymphocytes ↓ (≈ –73%)
0.34
• Number of neutrophils ↓ (≈ –52%)
0.34
• LPS-induced TNF-α production (Ex vivo, in vitro LPS-stimulation) ↑ 0.34
Exposure: oral (dietary)
(≈ +112% in peritoneal cavity, +103% in bone marrow, +14% (ns?) in
spleen)
Doses: 0, 0.001, 0.02 % (w/w)
• LPS-induced IL-6 levels (Ex vivo, in vitro LPS-stimulation) ↑ (≈ +33% 0.34
(equivalent to 0, 0.34, 4.3 mg/kg bw/d) in peritoneal cavity, +50% in bone marrow, +152% in spleen)
• LPS-induced TNF-α production (Ex vivo, in vivo LPS-stimulation) ↑ 0.34
(+126% in peritoneal cavity, +150% in bone marrow); ↓ (-73% in
spleen)
• LPS-induced IL-6 levels (Ex vivo, in vivo LPS-stimulation) ↑ (≈ +40% 0.34
in peritoneal cavity, +150% in bone marrow); ↓ (≈ –60% in spleen)
• Serum TNF-α levels after LPS injection ↑ (≈ +473%)
0.34
• Serum IL-6 levels without LPS injection ↑ (≈ +150%)
0.34
• Serum IL-6 levels with LPS injection ↑ (≈ +92%)
0.34
Body weight gain (ns)
10-day-study
Liver:
Mouse (C57BL/6 (H-2b))
• Rel. liver weight ↑ (≈ +67%)
• Histological alterations in structure of parenchymal cells
n/sex/group = 4-7 (m only)
• Hypertrophy of hepatocytes around central vein
• Larger cell surface in centrilobular hepatocytes
• Total number of intrahepatic immune cells ↑ (≈ +100%)
Exposure: oral (dietary)
• Total number of intrahepatic myeloid-, lymphoid-, and erythroidrelated cells ↑ (e.g. +64% for granulocytes, +118% for
Dose: 0, 0.002% (w/w) (equivalent to
macrophages, +267% for myeloid suppressor cells, +283% for
0.65 mg/kg bw/d)
erythroid progenitor TER119, +85% for NK cells, +70% for B cells,
+78% for Helper T cells)
Clinical chemistry:
• Serum ALP ↑ (≈ +45%)
• Serum total cholesterol ↓ (≈ –23%)
• Serum triglycerides (≈ –36%)

48

• kidney, nervous

4.3
4.3
4.3
4.3

Serum PFOA concentration
(Qazi et al.,
system,
after 10 days:
2009)
haematopoietic 0.02%: 152 ± 8.6 µg/ml (357 ±
system, thyroid, 20.8 µM)
endocrine
system, clinical
chemistry

4.3
4.3

4.3
4.3
4.3
4.3
• kidney, nervous

Serum PFOA concentration
system,
after 10 days:
haematopoietic 0.002%: 87.6 ± 2.1 µg/ml
system, thyroid, (211.6 ± 5.1 µM)
endocrine system

(Qazi et al.,
2010)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFOA (96%
purity)

10-day study (+10 days recovery)
Mouse (C57BL/6 (H-2b))
n/sex/group = 8 (m only)
Exposure: oral (dietary)

Body weight ↓ (≈ –27% at LOEL)
Liver:
• Rel. liver weight ↑ (≈ +73% at LOEL)
Immune system:
• Rel. thymus weight ↓ (≈ –79% at LOEL)
• Rel. spleen weight ↓ (≈ –44% at LOEL)
• Rel. epididymal fat weight ↓ (≈ –93% at LOEL)
• Number of myeloid cells in bone marrow ↓ (≈ –35% at LOEL)
• Number of bone marrow B-lymphoid cells ↓ (≈ –27% at LOEL)

0.75

7.5

–

0.4

0.75
0.75
0.75
0.75
0.4

7.5
7.5
7.5
7.5
0.75

Doses: 0, 0.001, 0.002%, 0.02 % (w/w)
(control ad libitum or restricted/pair- Remarks: Most effects not recovered after 10 additional days. Pairfed to 0.02%) (equivalent to 0, 0.4,
fed controls indicate that effects at 0.02% are likely due to reduced
0.75, 7.5 mg/kg bw/d)
food uptake
PFOA (no
Body weight (ns)
5-week
purity
Liver:
Mouse (C57BL/6 and BALB/c,
specified, but premature)
• Rel. liver weight (C57) ↑ (m/f, ≈ +67%/+66%)
supplier was
• Rel. liver weight (BALB/c) ↑ (m/f, ≈ +55%/+65%)
Sigma Aldrich
• Liver cholesterol (BALB/c) ↓ (m/f, ≈ –80/60% in BALB/c mice)
which today n/sex/group = 2x3
• PPARα gene ex (BALB/c) ↑ (m, ≈ +33%; f: ns)
sells 95%
Clinical chemistry
purity PFOA) Exposure: oral (dietary)
• Plasma cholesterol (C57) ↑ (m/f, ≈ +27/+62%)
• Plasma cholesterol (BALB/c) ↑ (m, ≈ +24%; f: ns)
Reproductive tissue
Doses: 0, 3.5 mg/kg diet (equivalent to • Cholesterol in mammary (C57) ↑ (f, ≈ +10%)
0.55 mg/kd bw/d at 4 weeks of age
• Cholesterol in ovary (C57) ↑ (f, ≈ +70%)
(males and females); at 10 weeks of
age, male mice consumed ca. 0.33 and Remark: This study concludes that PFOA effects may depend on diet
female mice ca. 0.44 mg PFOA/kg
composition (PFOA leads to higher cholesterol in blood)
bw/d)
PFOA
21-day study
(ammonium Mouse (ICR)
salt, 98%
purity)
n/sex/group = 10 (m only)
Exposure: oral (drinking water)
Doses: 0, 2, 10, 50, 250 ppm (mg/l
drinking water) (equivalent to 0.49,

Body weight gain ↓ (≈ –192% at LOEL)
Body weight ↓ (≈ –33% at LOEL, unclear at which day or whether
averaged over all days)
Liver:
• Rel. liver weight ↑ (≈ +27%)
• Liver ALT activity ↑ (≈ +189% at LOEL)
• Liver AST activity ↑ (≈ +230% at LOEL)
• Hepatic TNF-α gene ex ↓ (≈ –50% at LOEL)
• Hepatic IL-1β gene ex ↓ (≈ –60% at LOEL)
• Hepatic TGF-β gene ex ↑ (≈ +40% at LOEL)
Immune system:

49

• kidney, nervous

(Qazi et al.,
2012)

system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

• kidney, nervous

system,
haematopoietic
system, thyroid

PFOA levels in plasma in C57 (Rebholz et
male/female mice:
al., 2016)
Control: 2/28 ng/mL
PFOA exposed: 26.9/44.3
µg/mL
PFOA levels in plasma in
BALB/c mice:
Control: 5/86 ng/mL
PFOA-exposed: 28.2/35.6
µg/mL

2.64
2.64

17.63
17.63

0.49
2.64
2.64
17.63
2.64

0.49
2.64
17.63
17.63
47.21
17.63

• nervous system,

haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Son et al.,
2008; Son
et al.,
2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
2.64, 17.63, 47.21 mg/kg bw/d)

• Alterations of splenic T-lymphocyte populations (↓ in CD4-CD8-,

CD4-CD8+, and CD4+CD8+; ↑ in CD4+CD8-)
• Splenic CD4-CD8+ Lymphocytes ↓ (≈ –65% at LOEL)

–

0.49

17.63
2.64
17.63
17.63
2.64

47.21
17.63
47.21
47.21
17.63

• Alterations of thymic T-lymphocyte populations (↑ in CD4-CD8-,

CD4-CD8+; ↓ in CD4+CD8+)
• Splenic TNF-α gene ex ↑ (≈ +83% at LOEL)
• Splenic IL-1β gene ex ↑ (≈ +100% at LOEL)
• Splenic TGF-β gene ex ↑ (≈ +100% at LOEL)
• Splenic c-myc gene ex ↑ (≈ +150% at LOEL)
• Thymic c-myc gene ex ↑ (≈ +90% at LOEL)

PFOA

3-week study
Mouse (C57BL/6N)
n/sex/group = 7-8 (m)
Exposure: oral (dietary, high fat diet
(HFD))
Doses: 0 (pair-fed to PFOA group), 5
mg/kg bw/d

PFOA (96%)

7-day-study
Mouse (Balb/c)
n/sex/group = 3x5 (f)

Remark: increased ALT and AST suggest hepatotoxicity
Body weight ↓ (≈ –11%/–19% with normal/HF diet)
Liver:
• Abs. liver weight ↑ (≈ +136%/+254% with normal/HF diet)
• Rel. liver weight ↑ (≈ +197%/+213% with normal/HF diet)
• Liver triglyceride ↑ (≈ +70%/+80% with normal/HF diet)
Clinical chemistry:
• Plasma ALT ↑ (≈ +608%/+1173% with normal/HF diet)
• Plasma AST ↑ (≈ +61%/+112% with normal/HF diet)
• Plasma ALP ↑ (≈ +467%/+400% with normal/HF diet)
• Plasma FFAs ↑ (≈ +24% only under HFD)
Other effects:
• Abs. epididymal white adipose tissue ↓ (≈ –78%/–63% with
normal/high fat diet)
• Rel. epididymal white adipose tissue ↓ (≈ –72%/–55% with
normal/high fat diet)
• Abs. subcutaneous white adipose tissue ↓ (≈ –72%/–66% with
normal/high fat diet)
• Rel. subcutaneous white adipose tissue ↓ (≈ –65%/–59% with
normal/high fat diet)
Remark: more gene expression and metabolomics data available (not
listed here). PFOA effects stronger than HFD effects. PFOA and HFD
effects add up in liver metabolism. “(…)HFD increases the risk of
PFOA exposure (…)”
Body weight after 7 days ↓ (≈ –23%)
Body weight gain ↓ (≈ –12% in PFOA, +14% in controlà –186% in
body weight gain, data in graph do not fit data from table)
Liver:
• Rel. liver weight ↑ (≈ +32/+10% without/with RVB 300)

50

• Kidney, nervous

system, thyroid,
endocrine system

• Kidney, nervous

system,
haematopoietic
system, thyroid,
endocrine

(Tan et al.,
2013)

(Vetvicka
and
Vetvickova,
2013)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Exposure: oral (gavage, in PBS)
Doses: 0, 20 mg/kg bw/d

Immune system:
• Rel. spleen and thymus weight (ns)
• Thymic cellularity ↓ (≈ –47/–21% without/with RVB 300)
• T cell lymphocyte proliferation ↓ (≈ –22/–14% without/with RVB
300)
• B cell lymphocyte proliferation ↓ (≈ –14% only without RVB 300)
• Phagocytosis of peripheral neutrophils ↓ (≈ –57/–8% without/with
RVB 300)
• Splenic NK cell activity ↓ (≈ –64/–16% without/with RVB 300)

Remark: PFOS data available. Focus of study on combined treatment
of PFAS and RVB (Glucan-resveratrol-vitamin C), which basically
counteracts PFOA effects.
PFOA (purity 7-day-study+2, 5, or 10 days recovery Body weight ↓ (≈ –20%, recovery after 5 days)
was not
Liver
Mouse (C57BL/6)
specified, but
• Abs liver weight ↑ (≈ +90%, further increase during recovery:
Sigma Aldrich
+110% after 2 days, +130% after 5 days recovery, slow recovery
n/sex/group = 4 (m)
delivers in
after 10 days (+80%))
• Hepatic CPT activity ↑ (≈ +300%, slow recovery (down to +160%
95% purity)
after 10 days))
Exposure: oral (dietary)
• Hepatic COT activity ↑ (≈ +900%, slow recovery (down to +400%
after 10 days)
Doses:0, 0.02% (w/w) (equivalent to 24 Fat tissue
mg/kg bw/d)
• Epididymal adipose tissue ↓ (≈ –80%, recovery after 10 days)
• Retroperitoneal adipose tissue ↓ (≈ –90%, recovery after 5 days)
• LPL activity of epididymal adipose tissue ↓ (≈ –61%, recovery after 5
days)
Clinical chemistry
• Serum triglycerides ↓ (≈ –29%, even further reduction after 2 days
recovery (-57%), after 5 and 10 days recovery level comparable to
levels of treatment day 7
• Serum cholesterol ↑ (≈ +100% after 2 days recovery, then recovery
after 10 days)
Body weight ↓ (≈ –17%)
PFOA
10-day-study, mouse (C57BL/6)
Liver
• Abs./rel. liver weight ↑ (≈ +96%/+136)
n/sex/group = 4 (m)
Immune system:
• Abs. + rel. thymus weight ↓ (≈ –85%)
• Abs.+ rel. thymic DNA contet ↓ (≈ –39% for rel)
Exposure: oral (dietary)
• Abs. + rel. spleen weight ↓ (≈ –30%)
• Abs. splenic DNA content ↓ (≈ –49%)

51

system, clinical
chemistry

• Kidney, nervous

system,
haematopoietic
system, thyroid,
endocrine
system,

• Kidney, nervous

system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

(Xie et al.,
2003)

(Yang et
al., 2000)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 0.02% (w/w) (equivalent to
30 mg/kg bw/d)

• Thymocyte cellularity ↓ (≈ –56% for CD4-CD8-, -95% for CD4+CD8+,

-64% for CD4+, -72% for CD8+)
• Splenocyte cellularity ↓ (≈ –75% for CD3+, -78% for CD4+, -74% for

CD8+, -86% for CD19+)

PFOA

PFOA

PFOA

Remark: most effects already present after 5 days
Body weight ↓ (≈ –15% at 0.02% after 5 days, data not shown for
other doses)
Mouse (C57BL/6)
Liver:
• Rel. liver weight ↑ (≈ +60% at LOEL)
11.5
n/sex/group = 4 (m)
Immune system:
• Rel. thymus weight ↓ (≈ –50% at LOEL)
3.5
• Rel. spleen weight ↓ (≈ –35% at LOEL)
3.5
Exposure: oral (dietary)
• Hepatic acyl-CoA oxidase activity ↑ (≈ +300% with palmitoyl-CoA as –
substrate, +1000% with lauroyl-CoA as substrate, increase starts
Doses: 0, 0.001%, 0.003%, 0.01%,
already on day 1)
0.02%, 0.05% (w/w) (equivalent to 0, 1, • Total number of thymocytes ↓ (≈ –90% at 0.02% after 7 days)
3.5, 11.5, 23, 57.5 mg/kg bw/d)
• Thymocyte cellularity ↓ (≈ –96% for CD4+CD8+, –62% for CD4+, –
77% for CD8+, –67% for CD4-CD8-; recovery complete after 10 days)
• Splenocyte cellularity ↓ (≈ –73% for CD3+, –68% for CD4+, –63% for
CD8+, –75% for CD19+; recovery complete after 10 days)
Body weight ↓ (≈ –14% in WT)
7-day-study
Mouse (C57BL/6 (WT) or PPARα-null) Liver:
• Abs. liver weight ↑ (≈ +86%/+119% in WT/PPARα-null)
Immune system:
n/sex/group = 4 (m)
• Abs. spleen weight ↓ (≈ –39% in WT)
• Splenocyte number ↓ (≈ –78% in WT)
• Abs. thymus weight ↓ (≈ –79%/–39% in WT/ PPARα-null)
Exposure: oral (dietary)
• Thymocyte number ↓ (≈ –84%/–39% in WT/ PPARα-null)
• Hepatic Palmitoyl-CoA oxidase activity ↑ (≈ +1900% in WT)
Doses:0, 0.02% (w/w) (equivalent to • Thymocyte cellularity ↓ (≈ –93% for CD4+CD8+, –67% for CD4+CD833.3 mg/kg bw/d (WT) or 29.8 mg/kg , –68% for CD4-CD8+, –55% for CD4-CD8-)
bw/d (PPARα))
• Splenocyte cellularity ↓ (≈ –42% for CD3+, -85% for CD19+)
Immune system:
10-day-study, 2-10 days
• IgM ↓ (≈ –73%/-72% without/with HRBC- immunisation)
Mouse (C57BL/6)
• IgG1 ↓ (≈ –29%/–99.95% without/with HRBC-immunisation)
• IgG2b ↓ (≈ –50%/–95% without/with HRBC-immunisation)
n/sex/group = 4 (m)
• IgG3 ↓ (≈ –33%/–91% without/with HRBC-immunisation)
10-day-study, 2-10 days

Exposure: oral (dietary)

Remark: IgM only antibody almost recovered after 6 days, other

52

• Kidney, nervous

23
11.5
11.5
1

system,
haematopoietic
system, thyroid,
endocrine
system, clinical
chemistry

• kidney, nervous

system,
haematopoietic
system, immune
system,
endocrine
system, clinical
chemistry

• Body weight,

liver, kidney,
nervous system,
haematopoietic
system,
endocrine
system, clinical

(Yang et
al., 2001)

(Yang et
al., 2002b)

(Yang et
al., 2002a)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
antibodies: not fully recovered

chemistry

Doses: 0, 0.02% (w/w) (equivalent to
30 mg/kg bw/d)
PFOA
14 days-study + 14 days recovery
(ammonium Rabbit (albino)
salt)

Body weight decreased in 14 days of treatment (compared to intial
values) and increased again after 14 recovery days (males and
females; no control group)

(O’Malley
and Ebbins,
1981)

•

n/sex/group = 6-10
Exposure: dermal (percutaneous, 40%
of body surface area covered, 6h/day
(10 applications: 5 application days, 2
rest days, 5 application days, 2 days
rest, then 14 days recovery)
Dose: 100 mg/kg (no control group,
only compared to initial values…)
PFOA (100%) 12-days

Body weight (day 5) ↓ (≈ –13%)
Body weight (day 10) ↓ (≈ –15%)
20
Body weight (recovery day 14) ↓ (≈ –8%)
20
Liver
20
n/sex/group = 15 (m)
• Abs./rel. liver weight directly after last application ↑ (≈ +53%/+55%
at LOEL)
0
Exposure: Dermal (skin patch on back, • Abs./rel. liver weight after 14 days of recovery ↑ (≈ +33%/+54% at
LOEL)
20
15% of body surface), 6h/day (10
applications: 5 application days, 2 rest • Abs./rel. liver weight after 42 days of recovery ↑ (≈ +30%/+31% at
LOEL)
200
days, 5 application days)
Haematopoietic system:
• Red blood cell directly after last application ↑ (≈ +53%/+55% at
Doses: 0, 20, 200, 2000 mg/kg
LOEL)
Rat (Crl:CD)

Remarks: some treatment related toxicity resolved during a 42-day
recovery period (but not liver weight); dermal LD50 (7000 mg/kg for
males, 7500 mg/kg for females)

•

200
200
200
20
200
2000

Organofluorine concentrations (Kennedy,
in blood (ppm) after last
1985)
application:
0 mg/kg: 10.2 ± 5.5
20 mg/kg: 52.4 ± 3.7
200 mg/kg: 79.2 ± 29.9
2000 mg/kg: 117.8 ± 21.1
Organofluorine concentrations
in blood (ppm) after 14 days
recovery:
0 mg/kg: 2.7 ± 0.9
20 mg/kg: 9.5 ± 0.5
200 mg/kg: 26.2 ± 4.8
2000 mg/kg:44.8 ± 13.1
Organofluorine concentrations
in blood (ppm) after 42 days
recovery:
0 mg/kg: 0.5 ± 0.2
20 mg/kg: 1.2 ± 0.5
200 mg/kg: 3.7 ± 2.0

53

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
2000 mg/kg:8.2 ± 4.1
PFOA
Repeated-dose-inhalation
(ammonium Rat (Crl:CD)
salt, 100%)

Body weight (day 5) ↓ (≈ −7%)
8
Body weight (recovery day 8) ↑ (≈ +5% at MD)
Liver:
• Abs./rel. liver weight directly after last application ↑ (≈ +52%/+45%
n/sex/group = 24 (m)
at LOEL)
1
• Abs./rel. liver weight after 14 days of recovery ↑ (≈ +19%/+23% at
LOEL)
1/8
Exposure: inhalation, 6h/day (10
applications: 5 application days, 2 rest • Abs./rel. liver weight after 28 days of recovery ↑ (≈ +16%/+23% at
LOEL)
1/8
days, 5 application days)
Clinical chemistry:
• Serum ALP activity (after last application) ↑ (≈ +36% at LOEL)
1
Doses: 0, 1, 8, 84 mg/m³
• Serum ALP activity (after 14 days recovery) ↑ (≈ +38% at LOEL)
8

84

•

APFO concentrations in blood (Kennedy
(ppm) after last application: et al.,
1986)
0 mg/m³: 1.4
1 mg/m³: 13
8 mg/m³: 47
84 mg/m³: 108

8
8/84

Organofluorine concentrations
in blood (ppm) after 14 days
recovery:

8/84

0 mg/m³: 0.28
1 mg/m³: NA
8 mg/m³: NA
84 mg/m³: 10

8
84

Organofluorine concentrations
in blood (ppm) after 28 days
recovery:
0 mg/m³: 0.10
1 mg/m³: 1.2
8 mg/m³: 3.8
84 mg/m³: 7.1
Organofluorine concentrations
in blood (ppm) after 42 days
recovery:
0 mg/m³: 0.032
1 mg/m³: NA
8 mg/m³: NA
84 mg/m³: 1.8
Organofluorine concentrations
in blood (ppm) after 84 days
recovery:
0 mg/m³: 0.015
1 mg/m³: NA
8 mg/m³: NA
84 mg/m³: 0.84
PFOA (>98%) 28-day RDT study, NTP study, similar to Body weight ↓ (m, 5 mg/kg bw/d: ≈ –12%, 10 mg/kg bw/d: −19%)
OECD TG 407
Liver:
• Abs.+ rel. liver weight ↑ (m/f, ≈ +16/14% at LOAEL)

54

1.25/100

2.5/−

−/12.5

0.625/25

•

Although females were
(NTP,
administered a 10-fold higher 2019b)
dose of PFOA, males had a

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Rat (SD)

• Hepatocyte hypertrophy ↑

−/50
−

• Acetyl-CoA activity↑ (m, ≈ +222% at LOAEL)

n/sex/group = 10 (m+f)

0.625/100
0.625

higher plasma concentration
compared to females across
the dose groups (up to 1,000fold higher levels)

• Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 ↑ (≈ +246-

1971% at LOAEL)
−
0.625
Kidney:
• Abs./rel. kidney weight ↑ (m/f, ≈ +11/+7% at LOAEL)
−/25
0.625/50
Exposure: oral (gavage, in distilled
Immune system:
water)
• Abs. spleen weight ↓ (m, ≈ −12% at LOAEL; f: ns)
1.25
2.5
• Abs. thymus weight ↓ (m, ≈ −12% at LOAEL; f: ns)
5
10
Doses (m): 0, 0.625, 1.25, 2.5, 5, 10
Thyroid:
mg/kg bw/d
• Rel. thyroid weight ↑ (m, ≈ +17% at LOAEL; f: ns))
0.625
1.25
Doses (f): 0, 6.25, 12.5, 25, 50, 100
• Thyroid gland follicular cell hypertrophy
5/50
10/100
mg/kg bw/d
Haematopoietic system:
• Erythrocytes , haemoglobin, Haematocrit ↓ (m, ≈ −4-6% at LOAEL) 0.625
1.25
• Reticulocyte ↓ (m, ≈−15% at LOAEL)
−
0.625
Clinical chemistry:
• ALT ↑ (m, ≈ +19-47%)
−
0.625
• ALP ↑ (m, ≈ +13-89%)
−
0.625
• ALP ↑ (m, ≈ +22-38%)
1,25
2.5
• Cholesterol ↓ (m, ≈ -37%)
−
0.625
Endocrine system (m):
• T3 (≈ −40% at LOAEL), free T4 (≈ −79% at LOAEL), total T4 ↓ (≈ −91%
at LOAEL)
−/50
0.625/100
Reproductive Tissue (m only):
• L. Cauda Epididymis weight ↓ (m, ≈ -11% at LOAEL)
2.5
5
• L. Epididymis weight ↓ (m, ≈ -9% at LOAEL)
5
10
• Sperm (106/g cauda epididymis) ↓ (m, ≈ -24% at LOAEL)
5
10
Other effects:
• Hyperplasia/inflammation of respiratory epithelium
−/6.25
0.625/12.5
PFNA: Perfluorononanoic acid (Syn: Heptadecafluorononanoic Acid, CAS no: 375-95-1, EC no: 206-801-3, Mol. formula: C9HF17O2, MW: 464,xx?; registered under REACH, self classification available)
5-day RDT study, non-guideline study
PFNA,
Perfluoronona Rat (Wistar)
noic acid
(analytical
n/sex/group = 4
grade), EC:
206-801-3,
CAS: 375-95- Exposure: i.p. injection (vehicle:
1, C9HF17O2 propyleneglycol:water (1:1, v:v)

Liver:
• Peroxisomal β-oxidation ↑ (m/f)

• kidney, nervous

−/5

Remarks: Mechanistic study to assess the MoA; peroxisomal βoxidation is statistically highly correlated with PFCA concentration in
the liver (r=0.850, p<0.001) à internal dose in liver decisive of effect
not carbon chain length or sex

55

2.5/10

PFNA conc. in the liver at
system, immune highest dose:
system,
m: 358 ± 19 μg/g liver
haematopoietic
f: 102 ± 11 μg/g liver
system, thyroid,
endocrine system

(Kudo et
al., 2000)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d
PFCAs assessed: PFHpA, PFOA, PFNA,
PFDA
PFNA

5-day RDT study, non-guideline study Liver:
• Abs./rel. liver weight ↑
−/−
Mouse (ddy)
• Peroxisomal β-oxidation ↑ (m/f)
−/−
• Activity of hepatic microsomal 1-Acly-GPC acyltransferase (m, only −
n/sex/group = 3-5
20 mg/kg bw/d tested) ↑
Exposure: i.p. injection (vehicle:
propyleneglycol:water (1:1, v:v)

• kidney, nervous

2.5/2.5
2.5/2.5
20

C-length and sex dependent (Kudo et
system, immune accumulation of PFCAs in the al., 2006)
liver (higher concentrations in
system,
haematopoietic males and with higher C
system, thyroid, length)
endocrine system

Remarks: Mechanistic study to assess the MoA; hepatic peroxisomal
β-oxidation is statistically highly significantly correlated with PFCA
concentration in the liver (m: r=0.9201, p<0.001; f: r=0.9254,
p<0.001) à internal dose in liver decisive for effect

Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d
PFCAs assessed: PFHxA, PFHpA, PFOA,
PFNA
PFNA (97%)

14-days RDT study, non-guideline
Rat (SD)
n/sex/group = 6 (m)
Exposure: oral (gavage, in distilled
water)

Liver:

• kidney, nervous

• Hepatocytes with focal vacuolations ↑

1
−
−
1

5
0.2
0.2
5

• mRNA of G6PC/GLUT2 ↑

1
1
1

5
5
5

Clinical chemistry:
• Serum glucose ↑ (≈ +9% at LOEL)
• Serum HDL ↓ (≈ -18-55%)
• Serum LDL ↑ (≈ +100% at LOEL)

0.2
−
1

1
0.2
5

• mRNA levels of CYP4A1 and ACOX ↑
• Hepatic TNFα level ↑
• Lipid accumulation in liver ↑

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Fang et
al., 2012b)

Remarks: Mechanistic study to assess the MoA

Doses: 0, 0.2, 1, 5 mg/kg bw/d
PFNA (97%)

14-days RDT study, non-guideline
Rat (SD)
n/sex/group = 6 (m)
Exposure: oral (gavage, in distilled
water)
Doses: 0, 0.2, 1, 5 mg/kg bw/d

Liver:

• kidney, nervous

• MDA in liver ↑
• Glycogen in liver ↑

Remarks: Mechanistic study to assess the MoA: PFNA caused
oxidative stress in liver and inhibited hepatic insulin signal pathway,
accelerating the output of glucose and increasing glycogen synthesis

56

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Fang et
al., 2012a)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFNA (97%)

7-days RDT study, non-guideline
Rat (SD, diabetes induced via
streptozotocin)
n/sex/group = 10 (m)
Exposure: oral (gavage, in distilled
water)

in the rat liver
Liver:
• Hepatic cholesterol ↑ (≈ +91% at LOEL)
• Hepatic triglycerides ↑ (≈ +180% at LOEL)
• G6P-dehydrogenase activity ↑ (≈ +19% at LOEL)
• Hepatic lipase ↓ (≈ -68% at LOEL)
Clinical chemistry:
• Serum ALT ↑ (≈ +60% at LOEL)

• kidney, nervous

0.2
1
0.2
1

1
5
1
5

0.2

1

1

3

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Fang et
al., 2015)

Remarks: Mechanistic study to assess the MoA

Doses: 0, 0.2, 1, 5 mg/kg bw/d
PFNA (97%)

14-days RDT study, non-guideline
Mouse (BALB/c (SD))

Body weight↓ (≈ –15% at LOEL)
• Body weight gain ↓ (≈ –517%)

nervous system,
Clinical chemistry

Immune system:
n/sex/group = 6 (m)

• Abs. spleen weight ↓ (≈ –9.6% (text), ≈ –34% (graph) at LOEL)
• Rel. spleen weight ↓ (≈ –32%)
• Splenocyte apoptosis ↑ (≈ +90%)

Exposure: oral (gavage)

• Abs./rel. thymus weight ↓ (≈ –33.3%/–39% at LOEL)
• Thymocyte apoptosis ↑ (≈ –+67%)

Doses: 0, 1, 3, 5 mg/kg bw/d

• Liver, kidney,

• IL-4 in spleen homogenates ↓ (≈ –17%)
• IFN-γ in spleen homogenates ↓ (≈ –50%)

1
3
3
1
3
3

3
5
5
3
5
1
5

3
1
1

5
3
3

(Fang et
al., 2008)

• Thymic gene expression of PPAR-α ↑ (≈ +510%/6.1-fold at 1 mg/kg)
• Thymic gene expression of PPAR-γ ↑ (≈ +160%/2.6-fold at 1 mg/kg)
• Thymic gene expression of IL-1-γ ↑ (≈ +200%/3-fold at 1 mg/kg)

Endocrine system:
• ACTH ↑ (≈ +72% at LOEL)
• Cortisol ↑ (≈ +17% at LOEL)

PFNA (97%)

14-day-study
Rat (SD)
n/sex/group =6 (m)
Exposure: Oral (gavage)
Doses: 0, 1, 3, 5 mg/kg bw/d

Body weight ↓ (≈ –18% at LOEL)
Immune system:
• Abs. thymus weight ↓ (≈ –20% at LOEL)
• Rel. thymus weight ↓ (≈ –58% at LOEL)
• Thymocyte apoptosis ↑
• Serum IL-1 ↑ (≈ +215% at LOEL)
• Serum IL-2 ↓ (≈ –29% at LOEL)
• Serum IL-4 ↑ (≈ +106% at LOEL)
• Thymic PPARα gene ex. ↑ (≈ +120% at LOEL)
• Thymic PPARγ gene ex. ↑ (≈ +190% at LOEL)
Endocrine system:

57

1
3

3
5

1
1
3
1

3
3
5
1
3

• Liver, kidney,

nervous system,
Clinical chemistry

(Fang et
al., 2009)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)

PFNA (97%)

14-day-study
Rat (SD)
n/sex/group = 6 (m)
Exposure: oral (gavage)
Doses: 0, 1, 3, 5 mg/kg bw/d

PFNA (97%)

14-day-study
Rat (SD)
n/sex/group =6 (m)
Exposure: oral (gavage)

• Cortisol ↑ (≈ +67% at LOEL)

3

5

Remark: More data (mainly related to MAPK and NF-κB signalling
pathways) available
Feng 2009, 2010 and Fang 2009, 2010 probably all one experiment
and different endpoints published separately
Immune system
• Abs. spleen weight ↓ (≈ –22% at LOEL)
• Rel. spleen weight ↓ (≈ –8% at LOEL)
• Lymphoid cell apoptosis in spleen ↑
• Splenic IL-1 ↑ (≈ +49% at LOEL)
• Splenic IL-6 ↑ (≈ +51% at LOEL)
• Splenic TNF-α ↑ (≈ +30% at LOEL)
• Splenic IFNγ ↓ (≈ –35% at LOEL)
• Splenic IL-10 ↓ (≈ –57% at LOEL)
• Splenic H2O2 conc. ↑ (≈ +31% at LOEL)
• Splenic SOD activity ↓ (≈ –42% at LOEL)
• Splenic PPARα gene ex. ↑ (≈ +160% at LOEL)
• Splenic PPARγ gene ex. ↑ (≈ +130% at LOEL)

3
1
3
3
3
3
3
3
1
1
1

1
5
3
5
5
5
5
5
5
3
3
3

Remark: IL-1, IL-6, IFNγ, and TNF-α are pro-inflammatory, IL-10 is
anti-inflammatory. More protein expression data (by Western
blotting) available
Endocrine system
• Serum testosterone ↓ (≈ –85% at LOEL)
• Serum estradiol ↑ (≈ +104% at LOEL)
• Apoptotic cells in testes ↑ (≈ +367% at LOEL)
• Fas gene ex ↑ (≈ +90% at LOEL)
• Bax gene ex ↑ (≈ +36% at LOEL)
• Bcl-2 gene ex ↓ (≈ –27% at LOEL)

3
3
1
3
3
1

5
5
3
5
5
3

1
3
-

3
1
1
5
1

• Body weight,

liver, kidney,
nervous system,
endocrine
system, clinical
chemistry

(Fang et
al., 2010)

•

(Feng et
al., 2009)

•

(Feng et
al., 2010)

Doses: 0, 1, 3, 5 mg/kg bw/d
PFNA (97%)

14-day-study
Rat (SD)
n/sex/group =6 (m)

Reproductive tissue
• Vacuolization between Sertoli cells and spermatogonia ↑
• Testicular WT1 protein levels ↑ (≈ +59% at LOEL)
• Testicular transferrin protein levels ↓ (≈ –33% at LOEL)
• Serum MIS ↑ (≈ +22% at LOEL)
• Serum inhibin B ↓ (≈ –10% at LOEL)

Exposure: oral (gavage)

58

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 1, 3, 5 mg/kg bw/d
PFNA
14-day RDT studies
(ammonium Mouse (Crl:CD-1)
salt, 99%)
n/sex/group = 5

Remark: in vitro study with primary sertoli cells also available.
Overall, the study shows that PFNA treatment led to the damage of
specific secretory functions of Sertoli cells
Mortality ↑ (all mice at 300 and 3000 ppm died before end of study) 3.8
Body weight ↓ (data not shown)
1.3
Liver
• Abs. liver weight ↑ (m/f, ≈ +58/+56% at LOEL)
-/• Rel. liver weight ↑ (m/f, ≈ +48/+35% at LOEL)
-/-

38
3.8
0.38/0.38
0.38/0.38

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Kennedy,
1987)

Exposure: oral (dietary)
Doses: 0, 3, 10, 30, 300, 3000 ppm
(equivalent to 0, 0.38, 1.3, 3.8, 38, 380
mg/kg bw/d)
PFNA (97%)

14-days RDT study, non-guideline
Mouse (Balb/c)
n/sex/group = 8 (m)
Exposure: oral (gavage)
Doses: 0, 0.2, 1, 5 mg/kg bw/d

PFNA (purity 14-days RDT study, non-guideline
not specified) study
Rat (Wistar)

Body weight ↓ (≈ -29% at LOAEL)
Liver:
• Rel. liver weight ↑ (≈ +9−140%)
• Hepatic triglycerides ↑ (≈ +67% at LOEL)
• Hepatic cholesterol ↑ (≈ +18% at LOEL)
• mRNA of Cyp4A10/ACOX1 ↑ (≈ +% at LOEL)
Clinical chemistry:
• Serum ALT ↑ (≈ +275% at LOEL)
• Serum AST ↑ (≈ +185% at LOEL)
• Serum triglyceride ↓ (≈ -71% at LOEL)
• Serum cholesterol ↓ (≈ -33% at LOEL)

1

Remarks
Mechanistic study to assess the MoA
Body weight ↓ (≈ -31% at LOAEL)
Liver:
• Hepatic OATP4C1 protein ↓
Clinical chemistry:
• Serum corticosterone ↑

n/sex/group = 10 (m)
Exposure: oral (gavage)
Doses: 0, 0.0125, 0.25, 5 mg/kg bw/d

59

5

−
−
−
−

0.2
0.2
0.2
0.2

1
1
1
1

5
5
5
5

0.25

5

−

0.0125

−

0.0125

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

• kidney, nervous

(Wang et
al., 2015)

(Hadrup et
Serum levels (μg/ml):
system, immune at 0.0125 mg/kg bw/d: 0.396 al., 2016)
system,
at 0.25 mg/kg bw/d: 30
haematopoietic
system, thyroid, at 5 mg/kg bw/d: 602
endocrine system

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
PFNA (purity 3-day RDT study, non-guideline study Liver:
• Rel. liver weight ↑ (≈ +88%)
not specified) Mouse (C57BL/6)
• mRNA of Cyp4A10 and Cyp2b10 ↑
n/sex/group = 4

• kidney, nervous

−
−

20
20

−
−
−

10
10
10

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

Remarks
Mechanistic study to assess the MoA

(Abe et al.,
2017)

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Exposure: i.p. (vehicle: corn oil)
Doses: 0 , 20 mg/kg bw/d
PFNA

7-day RDT study, non-guideline study Liver:
• Abs. + rel. liver weight ↑ (≈ +120%)
Mouse (SV129)
• Hepatic triglyceride ↑ (≈ +260%)
• Hepatic lipid content ↑ (≈ +540%)
n/sex/group = 4 (m)
Remarks
Mechanistic study to assess the MoA of PFOA, PFNA, PFHxS
Exposure: oral (gavage)

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Das et al.,
2017)

Doses: 0 , 10 mg/kg bw/d
PFNA

Formatted: French (France)

7-day RDT study, non-guideline study Liver:
• Abs. + rel. liver weight ↑ (≈ +56%)
−
Mouse (129S1/SvlmJ)
Remarks
Mechanistic study to assess the MoA of PFOA, PFNA, PFHxS, PFOS:
n/sex/group = 4 (m)
gene expression profiles significantly similar to profiles from mouse
tissues exposed to agonists of the constitutive activated receptor
(CAR), estrogen receptor α (ERα), and PPARγ; PPARγ and ERα were
Exposure: oral (gavage)
activated by all four PFCAs in trans-activation assays from the
ToxCast screening program
Doses: 0 , 1, 3 mg/kg bw/d

2.5/6.25
PFNA (>98%) 28-day RDT study, NTP study, similar to Mortality ↑ (m/f, ≈80-90% at LOAEL)
Body weight ↓ (m/f, ≈ −17/−10% at LOAEL)
0.625/
OECD TG 407
Liver:
1.56
Rat (SD)
• Abs. + rel. liver weight ↑ (m/f, ≈ +23/+21% at LOAEL)
−
• Hepatocyte, hypertrophy ↑ (m, 70% incidence at LOAEL)
−/3.12
n/sex/group = 10
• Hepatocyte, cytoplasmic alteration ↑ (m, 100% incidence at LOAEL) −/−
• Hepatocyte necrosis ↑ (m, 50% incidence at LOAEL)
1.25/6.25
• Hepatocyte, cytoplasmic vacuolization (m, 60% incidence at LOAEL) 0.625
Exposure: oral (gavage, in distilled
• Acetyl-CoA activity ↑ (m, ≈ +391% at LOAEL)
−
water)
• Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 ↑ (≈ +216−

60

• kidney, nervous

1

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Rosen et
al., 2017)

Formatted: French (France)
5/12.5
1.25/3.12
0.625/1.56
0.625/6.25
0.625/1.56
2.5/12.5
1.25
0.625/1.56
0.625/1.56

•

Plasma concentrations
(NTP,
normalized to dose generally 2019b)
increased with dose in males
and were similar across the
available doses in females;
Normalized plasma
concentrations were generally
five- to nine-fold higher in
males compared to females

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses (m): 0, 0.625, 1.25, 2.5, 5, 10
mg/kg bw/d
Doses (f): 0, 1.56, 3.12, 6.25, 12.5, 25
mg/kg bw/d

1889% at LOAEL)
Kidney:
• Abs.+ rel. kidney weight ↑ (m/f, ≈ +15/+7% at LOAEL)
Immune system:
• Abs. spleen weight ↓ (m/f, ≈ −26/–11% at LOAEL)
• Rel. Spleen weight ↓ (m/f, ≈ −12/–11% at LOAEL)
• Abs. thymus weight ↓ (m, ≈ −31% at LOAEL; f: ns)
• Rel. thymus weight↓ (m, ≈ −18% at LOAEL; f: ns)
• Bone marrow hypocellularity
• Spleen atrophy
• Thymus atrophy
• Lymph node mandibular atrophy
• Lymph node mesenteric atrophy
Thyroid:
• Abs./rel. Thyroid weight ↑ (only m; f: NA)
Haematopoietic system (only m):
• Leukocytes , lymphocytes ↓ (m, ≈ −42/−51% at LOAEL)
• Reticulocyte ↓ (m, ≈−19% at LOAEL)
Endocrine system (m):
• Testosterone in m ↓ (≈ –81% at LOEL)
• Testosterone in f ↑ (≈ +30% at LOEL)
• Free T4 (≈ −75% at LOAEL), total T4 ↓ (≈ −91% at LOAEL)
• Thyroid stimulating hormone ↓ (≈ −46% at LOAEL)
Clinical chemistry:
• Total protein, globulin, albumin ↓
• Urea nitrogen ↑
• ALT ↑ (m, ≈ +48% at LOAEL)
• ALP ↑ (m, ≈ +92% at LOAEL)
• AST ↑ (m, ≈ +13% at LOAEL)
• Cholesterol ↓ (m, ≈ -26% at LOAEL)
• Triglycerides ↓ (m, ≈ -51% at LOAEL)
• Bile salts ↑ (m, ≈ +111% at LOAEL)
Reproductive Tissue (m only):
• L. Cauda Epididymis weight ↓ (m, ≈ -11% at LOAEL)
• L. Epididymis weight ↓ (m, ≈ -7% at LOAEL)
• Testis weight ↓ (m, ≈ -7% at LOAEL)
• Sperm (106/g cauda epididymis) ↓ (m, ≈ -18% at LOAEL)
• Germinal epithelium degeneration ↑
• Interstitial cell atrophy in testes ↑
• Seminiferous tubule spermatid retention ↑
Other effects:

61

−/−

0.625/1.56

0.625/1.56 1.25/3.12
0.625/3.12 1.25/6.25
0.625
1.25
0.625
1.25
1.25/6.25 2.5/12.5
2.5/6.25 5/12.5
1.25/6.25 2.5/12.5
2.5/6.25 5/12.5
2.5/6.25 5/12.5
1.25

2.5

1.25
−

2.5
0.625

1.25
−
−/1.56
0.625

2.5
1.56
0.625/3.12
1.25

−/−
0.625/1.56
−/1.56
0.625/3.12
0.625/1.56 1.25/3.12
0.625/1.56 1.25/3.12
0.625
1.25
−
0.625
−
0.625
−
0.625
0.625
−
0.625
0.625
1.25
1.25
1.25

1.25
0.625
1.25
1.25
2.5
2.5
2.5

Plasma concentrations after
28-d in m (μg/mL):
0.625 mg/kg bw/d: 57
1.25 mg/kg bw/d: 161
2.5 mg/kg bw/d: 380
5 mg/kg bw/d: 358
Liver concentrations after 28-d
in m (μg/mL):
0.625 mg/kg bw/d: 146
1.25 mg/kg bw/d: 249
2.5 mg/kg bw/d: 311
5 mg/kg bw/d: 313
Plasma concentrations after
28-d in f (μg/mL):
1.56 mg/kg bw/d: 26.4
3.12 mg/kg bw/d: 54.4
6.26 mg/kg bw/d: 112.2
12.5 mg/kg bw/d: too less
survivor

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
25
12.5
1.25/6.25 2.5/12.5
Forestomach: Epithelium hyperplasia, chronic inflammation ↑
PFDA: Perfluorodecanoic acid (Syn: Nonadecafluorodecanoic acid, CAS no: 335-76-2, EC no: 206-400-3, Mol. formula: C10HF19O2, MW: 514,xx?; registered under REACH, self classification available)
• Olfactory Epithelium Degeneration in f ↑

PFDA,
7-day RDT study, non-guideline study Body weight ↓
Perfluorodeca Rat (Wistar)
Liver:
noic acid
• Abs./rel. liver weight ↑
(purity not
• Peroxisomal β-oxidation ↑
specified), EC: n/sex/group = 4 (m)
• Microsomal I-acyl-GPC acyltransferase ↑
206-400-3,
• Cytosolic long-chain acyl-CoA hydrolase ↑
CAS: 335-76- Exposure: oral (diet)
• GSH S-transferase ↓
• Hepatic Triacylglycerol ↑
2, C10HF19O2
• Hepatic Cholesterol ↑
Doses: 0, 0.00125, 0.0025, 0.005 or
0.01% (equivalent to 0 , 1.5, 3, 6, 12
mg/kg bw/d)
PFDA
(analytical
grade)

6

5-day RDT study, non-guideline study Liver:
• Peroxisomal β-oxidation ↑ (m/f)
Rat (Wistar)
n/sex/group = 4
Exposure: i.p. injection (vehicle:
propyleneglycol:water (1:1, v:v)

12

−
1.5
−
1.5
3
−
6

1.5
3
1.5
3
6
1.5
12

2.5/2.5

5/5

−
−

20
20

1

2

Remarks
Mechanistic study to assess the MoA; peroxisomal β-oxidation is
statistically highly correlated with PFCA concentration in the liver
(r=0.850, p<0.001) à internal dose in liver decisive of effect not
carbon chain length or sex

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

PFDA conc. in the liver at
system, immune highest dose:
system,
m: 453 ± 19 μg/g liver
haematopoietic
f: 412 ± 33 μg/g liver
system, thyroid,
endocrine system

(Kawashim
a et al.,
1995)

• kidney, nervous

(Kudo et
al., 2000)

• kidney, nervous

(Abe et al.,
2017)

Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d
PFCAs assessed: PFHpA, PFOA, PFNA,
PFDA
PFDA (purity 3-day RDT study, non-guideline study Liver:
not specified) Mouse (C57BL/6)
• Rel. liver weight ↑ (≈ +55%)
• mRNA of Cyp4A10 ↑
n/sex/group = 4 (sex not reported)

Remarks
Mechanistic study to assess the MoA

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

Exposure: i.p. (vehicle: corn oil)
Doses: 0, 20 mg/kg bw/d
PFDA (97.3%) 28-day RDT study, NTP study to assess Body weight ↓ (≈ -22%)

62

• nervous system,

(Frawley et

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
immunotoxicity and hepatotoxicity
Rat (SD)
n/sex/group = 8 (f)
Exposure: oral (gavage)

Liver:
• Abs. liver weight ↑
• Rel. liver weight ↑
• Single cell hepatocyte necrosis (minimal severity) ↑
Kidney:
• Abs. kidney weight ↑
• Rel. kidney weight ↑
Haematopoietic system:
• Mean corpuscular haemoglobin ↓ (≈ −6% at LOAEL)

0.125
−
0.25

0.25
0.125
0.5

0.25
0.125

0.5
0.25

0.125

0.25

thyroid,
endocrine system

al., 2018)

Doses: 0, 0.125, 0.25, 0.5, 1, 2 mg/kg
bw/d
PFDA

PFDA (98%)

28-day RDT study, NTP study to assess LOAEL/NOAELs given in mg/kg bw/week
immunotoxicity and hepatotoxicity
Liver:
• Abs./rel. liver weight ↑
Mouse (B6C3F1/N)
Immune system:
• Rel. spleen weight ↑
n/sex/group = 8 (f)
• Total spleen cell numbers ↓ (≈ –24% at LOAEL)
• Number of B-cells ↓ (≈ –27% at LOAEL)
• Number of T-cells ↓ (≈ –22% at LOAEL)
Exposure: oral (gavage)
• Number of T-helper cells ↓ (≈ –19% at LOAEL)
• Number of cytotoxic T-lymphocytes ↓ (≈ –19% at LOAEL)
Doses: 0, 0.31, 0.625, 1.25, 2.5, 5
• Number of NK cells ↓ (≈ –18% at LOAEL)
mg/kg bw/week
• Number of macrophages ↓ (≈ –21% at LOAEL)
10-day-study
Mouse (C57B1/6)
n/Sex/group = 4 (m)
Exposure: oral (dietary)
Doses: 0, 0.02% (w/w) (equivalent to
24 mg/kg bw/d)

Body weight ↓ (≈ –32%)
Liver:
• Abs./rel. liver weight ↑ (≈ +36%/+100%)
• Hepatic protein content ↑ (≈ +24%)
• Hepatic mitochondrial protein content ↑ (≈ +298%)
• Hepatic cytosolic protein content ↓ (≈ –29%)
• Hepatic peroxisomal catalase specific/total activity ↓↑ (≈ –
58%/+66%)
• Hepatic lauroyl-CoA oxidase specific/total activity ↑
(≈ +257%/+1325%)
• Hepatic palmitoyl-CoA oxidation specific/total activity ↑
(≈ +442%/+1908%)
• Microsomal cytochrome P450 reductase ↑ (≈ +251%)
• Microsomal epoxide hydrolase ↑ (≈ +125%)
• Hepatic cytosolic DT-diaphorase ↑ (≈ +149%)
• Hepatic cytosolic glutathione peroxidase ↓ (≈ –36%)
• Hepatic cytosolic SOD ↑ (≈ +59%)

63

• nervous system,

0.31

0.625

0.625
2.5
2.5
0.625
0.625
0.31
0.625
0.31

1.25
5
5
1.25
1.25
0.625
1.25
0.625

thyroid,
endocrine system

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Frawley et
al., 2018)

(Permadi
et al.,
1992;
Permadi et
al., 1993)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Hepatic cytosolic epoxide hydrolase ↑ (≈ +144%)

PFDA
96%

10 day, non-guideline ReproTox study Gd 10-13:
• Maternal body weight gain ↓ (≈ –61% at LOEL)
Mouse (C57BL/6N)
• Abs. liver weight ↑ (≈ +10% at LOEL)
• Rel. liver weight ↑ (≈ +3% at LOEL)
n/sex/group = 10-14
• Gd 6-15:
• Maternal body weight gain ↓ (≈ –92% at LOEL)
Exposure: oral (gavage, in corn oil)
• Abs./rel. liver weight ↑ (≈ +61%/+127% at LOEL)

• kidney, nervous

8
0.5
0.25

16
1
0.5

3
0.3

6.4
1

(Harris and
Birnbaum,
1989)

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

Doses: 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32
mg/kg bw/day on gestation days (GD)
10-13 (4 consecutive days)
or 0, 0.03, 0.1, 0.3, 1, 3, 6.4, or 12.8
mg/kg bw/day, on GD 6-15 (10
consecutive days)
PFDA (>98%) 28-day RDT study, NTP study, similar to Body weight ↓ (m: ≈ –21–38%, f: ≈ −12–36% at LOAEL)
Liver:
OECD TG 407
• Abs./rel. liver weight ↑ (≈ +11/12% in m/f at LOAEL)
Rat (SD)
• Hepatocyte, hypertrophy ↑ (≈ 80-100% incidence at LOAEL)
• Hepatocyte, cytoplasmic alteration ↑ (80-100% incidence at
n/sex/group = 10
LOAEL)
• Hepatocyte necrosis ↑ (f only, 40% incidence at LOAEL)
• Hepatocyte, cytoplasmic vacuolization (90-100% incidence at
Exposure: oral (gavage, in distilled
LOAEL)
water)
• Acetyl-CoA activity ↑ (m only, ≈ +22-404%)
• Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 ↑
Doses: males: 0, 0.156, 0.312, 0.625, Kidney:
1.25, or 2.5 mg/kg bw/d
• Abs./rel. kidney weight ↑ (≈ +8–15% at LOAEL)
Immune system:
• Abs. spleen weight ↓ (m/f, ≈ –26/–36% at LOAEL)
• Rel. spleen weight ↓ (m/f, ≈ −19/–9% at LOAEL)
• Abs.thymus weight ↓ (m/f, ≈ –44/–65% at LOAEL)
• Rel. thymus weight ↓ (m/f, ≈ –29/–46% at LOAEL)
• Bone marrow hypocellularity (100% incidence at LOAEL)
• Thymus atrophy (80-90% incidence at LOAEL)
Thyroid:
• Abs.+rel. thyroid weight ↑ (m/f, ≈ +27–45%)
Haematopoietic system (only in m determined):
• Reticulocytes ↓ (≈−54–62% at LOAEL)

64

0.625/0.625 1.25/1.25
−/3.12
0.125/0.125
0.625/0.625 1.25/1.25
0.325/0.325 0.625/0.625
1.25
2.5
0.625/1.25
−
−/−

1.25/2.5
0.156
0.156/0.156

0.312/0.156 0.625/0.312
0.625
1.25/0.312
0.625/1.25
0.625/1.25
1.25/1.25
1.25/1.25

1.25
2.5/0.625
1.25/2.5
1.25/2.5
2.5/2.5
2.5/2.5

0.625/0.156 1.25/0.312

•

Plasma concentrations were (NTP,
higher in females (30% or
2019b)
less); normalized to dose
plasma concentrations
increased with dose in males
and females; Normalized
plasma concentrations
increased with dose, 1.9-fold
increase in males and a 1.4fold increase in females from
lowest to highest dose; Liver
concentrations in males
increased with dose, but when
normalized to dose
(μM/mol/kg), values
decreased with increasing
dose
Plasma concentrations after
28-d in m (μg/mL):
0.156 mg/kg bw/d: 8.5
0.312 mg/kg bw/d: 23

0.625/0.625 1.25/1.25

0.625 mg/kg bw/d: 47.7

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Erythrocytes ↑ (≈ +13–23% at LOAEL)

1.25/0.625
0.312/1.25

• Mean cell haemoglobin ↑

2.5/1.25
0.625/2.5

1.25 mg/kg bw/d: 101.6

2.5 mg/kg bw/d: 259.4
Endocrine system (m):
Liver concentrations after 28-d
• Adrenal gland weight abs. ↓ (≈ −15% (m) to ≈ −36% (f) at
−/1.25
0.156/2.5
in m (μg/mL):
LOAEL)
0.156 mg/kg bw/d: 44.7
• Testosterone in m ↓ (≈ −64% at LOAEL)
0.625
1.25
• Testosterone in f ↑ (≈ +32-355%)
0.156
0.312
0.312 mg/kg bw/d: 87.2
• free T4 ↓ (≈ −39-42% at LOAEL)
−/1.56
0.625/3.12
0.625 mg/kg bw/d: 163.9
• T3, female only ↑ (≈ +24% at LOAEL)
0.156/0.625 0.312/1.25
1.25 mg/kg bw/d: 308.8
Clinical chemistry:
2.5 mg/kg bw/d: 403.6
• Globulin (g/dL) ↓ (≈−14–22% at LOAEL)
−/0.156
0.156/0.312
• Urea nitrogen ↑ (≈ +25–38% at LOAEL)
0.625/0.625 1.25/1.25
• Glucose ↓ (≈−31% at LOAEL)
0.625/0.625 1.25/1.25
Plasma concentrations after
• ALT ↑ (≈ +44–45% at LOAEL)
0.156/0.625 0.312/1.25
28-d in f (μg/mL):
• ALP ↑ (≈ +22–35% at LOAEL)
0.156/0.156 0.312/0.312
0.156 mg/kg bw/d: 11.2
• AST ↑ (m, ≈ +14–30% at LOAEL)
−/0.625
0.156/1.25
• Cholesterol ↓ (≈−25–34% at LOAEL)
−/1.25
0.156/2.5
0.312 mg/kg bw/d: 25.7
• Triglycerides ↓ (m only, ≈ -36% at LOAEL)
0.625
1.25
0.625 mg/kg bw/d: 50.3
• Bile salts ↑ (m, ≈ +205–440% at LOAEL)
0.625/0.625 1.25/1.25
1.25 mg/kg bw/d: 117.2
Reproductive Tissue (m only):
2.5 mg/kg bw/d: 246.9
• L. Cauda Epididymis weight ↓ (m, ≈ -11% at LOAEL)
0.625
1.25
• L. Epididymis weight ↓ (m, ≈ -10% at LOAEL)
0.625
1.25
• Testis weight ↓ (m, ≈ -11% at LOAEL)
1.25
2.5
• Sperm (106/g cauda epididymis) ↓ (m, ≈ -30% at LOAEL)
1.25
2.5
• Interstitial cell atrophy in testes ↑ (80% incidence at LOAEL) 0.625
1.25
PFUnDA: Perfluoroundecanoic acid (Syn: Henicosafluoroundecanoic acid, CAS no: 2058-94-8, EC no: 218-165-4, Mol. formula: C11HF21O2, MW: 564,xx?; SVHC, candidate for authorisation under REACH, self
classification available)
42-day RDT study with the
PFUnDA
Liver:
Reproduction/Developmental Toxicity • Abs. liver weight ↑
(98.5%),
Perfluorounde Screening Test, according to OECD TG • Rel. liver weight ↑
canoic acid, 422
• Hepatocyte hypertrophy ↑ (≈58-52% incidence at LOAEL)
EC: 218-165-4,Rat Crl:CD (SD)
Immune system:
CAS: 2058-94• Abs./rel. spleen weight ↓ (m only, ≈ −19–23% at LOAEL)
Clinical chemistry:
8, C11HF21O2
n/sex/group = 12
• Serum ALP, ALT ↑ (m only, ≈ +26% for ALT and +139% for ALP)
• Serum urea nitrogen ↑ (≈ +46-61% at LOAEL)
Exposure: oral (gavage, vehicle: corn • Serum albumin ↓ (m only, ≈ −7% at LOAEL)
• Total protein ↓ (≈ −10-11% at LOAEL)
oil)

65

• nervous system,

0.3/0.3
0.1/0.3
0.3/0.3

1/1
0.3/1
1/1

0.3/0.3

1/1

0.3
0.3/0.3
0.3
0.3/0.3

1
1/1
1
1/1

endocrine system

(Takahashi
et al.,
2014)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 0.1, 0.3, 1.0 mg/kg bw/d
PFDoDA: Perfluorododecanoic acid (Syn: Tricosafluorododecanoic acid, CAS no: 307-55-1, EC no: 206-203-2, Mol. formula: C12HF23O2, MW: 614.10; SVHC substance, candidate list for authorisation, self
class.available)
PFDoDA,
Liver:
14-day RDT study
Perfluorodode Rat (SD)
• Abs. liver weight ↓ (≈ −19-26%)
canoic acid
• Rel. liver weight ↑ (≈ +25-30% at LOAEL)
(>99%), EC:
• Hepatic triglyceride ↑ (≈ +27% at LOAEL)
n/sex/group
=
6
(m)
206-203-2,
• Hepatic SOD/CAT activity ↑
CAS: 307-55• Hepatic mRNA of PPARα/γ, ACOX, CPT1, Cyp4A1, LDLR ↑
1, C12HF23O2 Exposure: oral (gavage), vehicle: 0.5% • Hepatic content of cholesterol ↑ (≈ +39% at LOAEL)
Clinical chemistry:
Tween-20)
• Serum triglyceride ↑ (≈ +96% at LOAEL)

• kidney, nervous

1
1
5
−
−
5

5
5
10
1
1
10

5

10

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Zhang et
al., 2008)

Doses: 0, 1, 5, 10 mg/kg bw/d
PFDoDA
(95%)

PFDoDA
(95%)

110-day RDT study

Body weight ↓ (≈ −7% at LOAEL)
0.2
Liver:
• Abs./rel. liver weight ↑ (≈ +10-18%)
−
• Lipid droplets and widespread disintegrated cell systems
0.02
n/sex/group = 10 (m)
• Swollen and vacuolated hepatocytes
0.05
• Hepatic mRNA of PPARα, CYP4A1, ACOX, Cd36
0.05
Exposure: oral (gavage), vehicle: 0.2% • Hepatic triglycerides ↑
Clinical chemistry:
Tween-20)
• Serum ALP ↑ (m only, ≈ +73% at LOAEL)
0.05
• Serum urea nitrogen ↑ (≈ +17% at LOAEL)
0.05
Doses: 0, 0.02, 0.05, 0.2, 0.5 mg/kg
• Serum glucose ↑ (≈ +9% at LOAEL)
−
bw/d
• Serum albumin ↑ (≈ +6% at LOAEL)
−
• Creatine kinase ↑ (≈ +23% at LOAEL)
−
• Serum lipoprotein levels ↓ (no quantification possible because data
are expressed as ratio derived from NMR spectra, no statistics to
determine LOAEL)
Kidney:
110-day RDT study
• Renal protein level of pyruvate carboxylase ↑ (≈ 10-18%)
−
Rat (SD)
• Renal protein level of isovaleryl coenzyme A dehydrogenase, malate
dehydrogenase 1 and dihydrolipoamide S-acetyltransferase ↑
0.05
n/sex/group = 6 (m)
Remarks
Exposure: oral (gavage), vehicle: 0.2% Study to assess MOA of nephrotoxicity: disorders in glucose and
amino acid metabolism may contribute to nephrotoxicity; α2u
Tween-20)
globulin may play a role in protecting the kidneys from PFDoDA
toxicity
Rat (SD)

66

0.5
0.02
0.05
0.2
0.2

• kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Ding et al.,
2009)

0.2
0.2
0.02
0.02
0.02

• liver, nervous

0.05
0.2

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

(Zhang et
al., 2011)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
Doses: 0, 0.05, 0.2, 0.5 mg/kg bw/d
PFDoDA
(purity not
specified)

110-day RDT study
Rat (SD)
n/sex/group = 6 (m)

Liver:
• Hepatic cholesterol ↑
−
• Hepatic triglycerides ↑
−
• Altered hepatic levels of signal transduction proteins (e.g. glycogen
synthase kinase, insulin receptor substrate)
−

• Kidney, nervous

0.2
0.2

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

0.2

(Zhang et
al., 2013)

Exposure: oral (gavage), vehicle: 0.2% Remarks
Study to assess MOA of hepatotoxicity: chronic PFDoDA exposure
Tween-20)
may inhibit insulin signal pathways and inhibition of GSK3 might
contribute to the observed increases of lipid levels in the liver
Doses: 0, 0.2, 0.5 mg/kg bw/d
PFDoDA
(97%)

PFDoDA

42-day RDT study, OECD TG 422, 14
day recovery (0, 2.5 mg/kg bw/d)

Body weight ↓ (≈ −33% at LOAEL)
0.5/0.5
Liver:
• Rel. liver weight ↑ (≈ +15-20% at LOAEL)
0.1/0.1
Rat (SD)
• Hepatic hypertrophy ↑
0.5
• Hepatic necrosis ↑
0.5
n/sex/group = 7
• Inflammatory cholestasis ↑
0.5
Kidney:
• Rel. kidney weight ↑, abs. ↓ (m only)
0.5
Exposure: oral (gavage)
Immune system:
• Abs. spleen weight ↓ ( ≈ −14–38% at LOAEL)
0.5/0.1
Doses: 0, 0.1, 0.5, 2.5 mg/kg bw/d (14- • White blood cells in recovery group ↓ (m only, ≈ −20%)
−
day recovery group: 0, 2.5 mg/kg
Thyroid:
bw/d)
• Abs. thyroid weight ↓ (m, ≈ −55% at LOAEL)
0.5
Haematopoietic system:
• Reticulocytes ↓ (m only, ≈ −63% at LOAEL)
0.5
• Mean corpuscular haemoglobin concentration ↑ (m only, ≈ +4%)
0.5
Clinical chemistry:
• Serum ALP ↑ (m only, ≈ +54% at LOAEL)
0.1
• Serum total cholesterol ↓ (m only, ≈ −33% at LOAEL)
−
• Serum glucose ↓ (m, ≈ −27% at LOAEL)
0.5
• Globulin-α2 ↓ (m, ≈ −14% at LOAEL)
0.1
Other effects:
• Pancreatic zymogen granules ↓ (m only, 5/7 animals at HD/LOAEL, 0.5
1/5 in recovery group, compared to 0/5-12 in control groups)
Reproductive Tissue (m only):
• Abs. L. Epididymis weight ↓ (m, ≈ −32% at LOAEL)
0.5
• Spermatid and spermatozoa counts ↓
0.5
110 day RDT study
Body weight ↓ (≈ –5.5% at LOEL)
0.2

67

2.5/2.5

•

(Kato et al.,
2015)

• Kidney, nervous

(Shi et al.,

0.5/0.5
2.5
2.5
2.5
2.5
2.5/0.5
2.5
2.5
2.5
2.5
0.5
0.1
2.5
0.5
2.5

2.5
2.5
0.5

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
95%

PFDoDA
95%

PFDoDA

Rat (SD)

Endocrine system:
• Serum testosterone ↓ (≈ –44% at LOEL)
Reproductive tissue:
n/sex/group = 6 (m)
• Cast-off cells in some seminiferous tubules in testes (no quant)
• Testicular gene expression of StAR ↓ (≈ –35% at LOEL)
• Testicular gene expression of P450scc ↓ (only at 0.05)
Exposure: oral (vehicle: Tween-20)
• Testicular gene expression of IGF-I ↓ (≈ –25% at LOEL)
• Testicular gene expression of IGF-IR and IL-1α ↓ (≈ –25%/-30% at
Doses: 0.02, 0.05, 0.2, 0.5 mg/kg bw/d LOEL)
• Testicular gene expression of GnRH-R and FSH ↓ (≈ –45% at LOEL)
• Testicular gene expression of LH ↓ (≈ –50%, only at 0.05mg/kg
bw/d)
Body weight ↓ (≈ –25% at LOEL)
14-days RDT
Clinical chemistry:
Rat (SD)
• Total serum cholesterol ↑ (≈ +38.5% at LOEL)
Endocrine system:
n/sex/group = 6 (m)
• Serum LH ↓ (≈ –30% at LOEL)(FSH ns)
• Testosterone ↓ (≈ –50% at LOEL)
• Estradiol ↓ (only at 5 mg/kg bw/d, ≈ –50%)
Exposure: oral (gavage, vehicle:
Reproductive tissue:
Tween-20)
• Apoptotic features present in Leydig cells, Sertoli cells and
spermatogenic cells
Doses: 1, 5, 10 mg /kg bw/d
• Abs. testis weight ↓ (≈ –22% at LOEL)
• Rel. testis weight ↑ (≈ +36% at LOEL)
• Testicular gene expression of SR-B1 ↓ (≈ –60% at LOEL)
• Testicular gene expression of StAR ↓ (≈ –50% at LOEL)
• Testicular gene expression of P450scc ↓ (≈ –90% at LOEL)
Liver:
110-day RDT study
• Hepatic SOD activity ↑
Rat (SD)
• TBARS in liver ↑
• Hepatic GPX activity ↑
n/sex/group = 4-10 (m)
• mRNA of PPARα/Cyp4A1↑
• mRNA of mitochondrial acyl-CoA-thioesterase 1 and hydroxyacylExposure: oral (gavage), vehicle: 0.2% CoA-dehydrogenase ↑

0.05

0.2

0.2
-

0.5
0.02

0.02
0.05

0.05
0.2

0.2

0.5

1

5

5

10

5
1

10
5

5

10

5
1
1
1
1

10
5
5
5
5

0.2
0.2
0.2
0.05

0.5
0.5
0.5
0.2

−

0.05

system, immune
system,
haematopoietic
system, thyroid

• Kidney, nervous

system, immune
system,
haematopoietic
system, thyroid

• Kidney, nervous

system, immune
system,
haematopoietic
system, thyroid,
endocrine system

2009)

(Shi et al.,
2007)

(Liu et al.,
2016)

Tween-20)
Doses: 0, 0.05, 0.2, 0.5 mg/kg bw/d
PFTrDA: Perfluorotridecanoic acid (Syn: Pentacosafluorotridecanoic acid, CAS no: 72629-94-8, EC no: 276-745-2, Mol. Formula: C13HF25O2, MW: 664.11; SVHC, candidate list for authorisation, no self class.
available )

68

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
•

PFTeDA: Perfluorotetradecanoic acid (Syn: Heptacosafluorotetradecanoic acid, CAS no: 376-06-7, EC no: 206-803-4, Mol. formula: C14HF27O2, MW: 714.11; SVHC, candidate list for authorisation; Self class.
available, but only Skin Corr. 1B)
PFTeDA,
42-day repeated dose and
Perfluorotetra reproductive/developmental toxicity
decanoic acid (OECD TG 422), 14-day recovery

Body weight:
• Body weight in RDT study ↓ (m, ≈ –7% only in recovery group, f: ns)
• Body weight in Repro/DevTox study ↓ (f, ≈ –6% at LOAEL on day 4 1
of lactation)
Rat (SD)
• Body weight ↓ (f, ≈ –9% at LOAEL throughout entire lactation
3
period)
n/sex/group = 12 (m+f)
• Food consumption ↓ (f, day 5 and 10 gestation; day 4 lactation)
3
Liver:
1
Exposure: oral (vehicle control,0.5% • Abs. liver weight ↑ (m, ≈ +22% at LOAEL, also in recovery group)
• Rel. liver weight ↑ (m/f, ≈ +19/+11% at LOAEL, also in recovery
1/3
carboxymethylcellulose sodium in
groups)
water)
• Centrilobular hepatocyte hypertrophy ↑ (m/f, also in recovery
1/3
Dose groups: 0, 1, 3, 10 mg/kg bw/day groups)
• Microgranulomas ↑ (f, also in recovery group; m: microgranulomas –
in treatment and control group)
Dosing regime
• Focal necrosis in one female
All animals: 14 day pre mating
Kidney:
Males: 42 day
• Abs. + rel. kidney weight (m/f, ns)
Females: during lactation up to 5 days Immune system:
after parturition (app. 42 days i.e. 14 • Thymic cortex atrophy ↑ (m: NA; f: at LOAEL)
3
premating, 1-2 days mating 21-23
• Abs.+rel. thymus weight (m/f, ns)
gestation, plus 5 days)
• Abs.+rel. spleen weight (m/f, ns)
• White blood cell count (m/f, ns)
Thyroid:
Recovery Group: high dose for 42 days
• Follicular cell hypertrophy ↑ (m; f: NA)
1
without mating and vehicle control,
Haematopoietic system:
followed by a 14 day recovery period
• Activated partial thromboplastin time ↓ (>10%)
–
• Females: decrease in extramedullary haematopoiesis
1
• Haemoglobin, haematocrit ↓ (f, both ≈ –8%, only in recovery group)
• Shortened prothrombin time ↓ (f, both ≈ –7%, only in recovery
group)
Clinical Chemistry:
• β-globulin protein fraction ↓ (m/f, ≈ –8/–13% at LOAEL)
3/3
• Serum ALP ↑ (m, ≈ +43% at LOAEL, +41% in recovery group, f: ns)
3
• BUN ↑ (m, ≈ +39% at LOAEL, not in recovery group; f: only in
3
recovery)
• Serum chloride ↑ (f, ≈ +3% at LOAEL, not in recovery group, m: ns) 3

69

• All major organs

3
10
10
3
3/10
3/10
10

10

3
10
3

10/10
10
10
10

and tissues have
been included, as
listed in the
manuscript

(HirataKoizumi et
al., 2015)

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Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
• Serum inorganic phosphorus ↓ (m, ≈ +8% only in recovery group; f:

ns)

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• Serum triglycerides ↓ (m, ≈ –46% only in recovery group; f: ns)
• Serum cholesterol ↓ (m: ns; f: ≈ –25% only in recovery group)

Other effects:
• Abs. weight pituitary gland ↓ (m)
1
3
• Hindlimb grip strength ↓ (m, ≈ –20-25%)
3
10
• Urinary parameters (ns)
Reproductive/developmental toxicity:
• Body weights in male and female pups ↓
3
10
• No effects on estrous cycle, copulation index, fertility index,
gestation length, Corpora lutea, implantation sites, delivered pubs,
sex ratio
PFPeDA: Perfluoropentadecanoic acid (Syn: Hexacosafluoro-13-(trifluoromethyl)tetradecanoic acid, CAS no: 18024-09-4, EC no: 241-936-1 , Mol. Formula: C15HF29O2 , MW: 764.12; no data on
ECHA chem search
•

Formatted: Spanish (Spain)

PFHxDA: Perfluorohexadecanoic acid (Syn: Perfluoropalmitic acid, CAS no: 67905-19-5, EC no: 267-638-1, Mol. formula: C16HF31O2, MW: 814.13; only self class. Skin Corr. 1B , no further ECHA data)
PFHxDA,
42-day Repeated dose and
Perfluorohexa reproductive/developmental toxicity
decanoic acid (OECD TG 422), 14-day recovery

Body weight:
• Body weights ↓ (m, ≈ –4% at LOAEL on day 35 and 42; f: ns)
20
• Food consumption ↓ (m in RDT experiment: at day 14 recovery
20
period; f in DevTox experiment: day 5 - 10 gestation; day 4 lactation)
Rat (SD)
Liver:
• Abs. liver weights ↑ (m, ≈ +19% at LOAEL, also in recovery group; f: 20
n/sex/group = 12 (m+f)
ns)
Exposure: oral (vehicle control,0.5% • Rel. liver weights ↑ (m, ≈ +30% at LOAEL, also in recovery group; f: 20
ns)
carboxymethylcellulose sodium in
• Centrilobular hepatocellular hypertrophy ↑ (m/f, m also in recovery 20
water)
group)
• Centrilobular fatty changes
20
Dose groups: 0, 4, 20, 100 mg/kg
Kidney:
bw/day)
• Abs. + rel. kidney weight (m/f, ns)
Immune system:
• Abs.+rel. thymus weight (m/f, ns)
Dosing regime:
• Abs.+rel. spleen weight (m/f, ns)
All animals: 14 day pre mating
• White blood cell count (m/f, ns)
Males: 42 day
Thyroid:
Females: during lactation up to 5 days • Rel. thyroid weight ↑ (m, +26% at LOAEL, not in recovery group; f: 4
after parturition
ns)
Endocrine system:
High dose recovery groups (14 days

70

•

100
100

100
100
100
100

20

Formatted: Spanish (Spain)

(HirataKoizumi et
al., 2015)

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
NO(A)EL
Substance study design, species, route of
Observed effects
(mg/kg
(%), EC/CAS, exposure, doses (guideline/similar
formula
to guideline/non-guideline)
bw/d)
Remarks
• T3 4, (m: ns; f, . —17% at LOAEL, not in recovery)
recovery after treatment)
.T4 4, (m, .-. —24% only in recovery group; f: ns)
Haematopoietic system (ns)
Clinical Chemistry
• Serum total bilirubin 1, (m, =-31% only in recovery group; f, =-25%20
at LOAEL, also in recovery group)
• BUN is (m: ns; f, =+21% at LOAEL, not in recovery group
20
• Serum sodium '1' (m: ns; f, =+1% at LOAEL, not in recovery group) 20
• Serum chloride T (m/f, :-. +3/+2% at LOAEL, f also in recovery group)20/4
• Serum cholesterol (m/f, ns)
• Serum triglycerides (m/f, ns)
Other effects
• Hindlimb grip strength 4, (m/f, =-20/-23%, only in recovery group)
• Urinary parameters (ns)
•Abs. + rel. adrenal weights 4, (m,-18-22% only in recovery group; f:
ns)
Reproductive/developmental toxicity
• Slightly lower body weights in male and female pups, no other
effects are reported

LO(A)EL
(mg/kg

Key parameters Serum/tissue concentrate- Reference
/ targets not
ion of PFAS /metabolites

bw/d)
4

addressed

(time of sampling)

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100
100
100
100/20

PFHpDA: Perfluoroheptadecanoic acid (Syn:, CAS no: EC no: , Mol. formula: C17HF33O2, MW: )
PFODA: Perfluorooctadecanoic acid (Syn: Perfluorostearic acid, CAS no: 16517-11-6, EC no: 240-582-5, Mol. formula: CHHF35O2, MW: 914.15; only self class. Skin Corr. 1B , no further ECHA data)
PFODA,
42-day Repeated dose and
Perfluoroocta reproductive/developmental toxicity
decanoic acid (OECD TG 422)
Rat (SD)
n/sex/group = 12 (m+f)

Exposure: oral (gavage; vehicle
control,0.5% carboxymethylcellulose
sodium in water)
Dose groups: 0, 40, 200, 1000 mg/kg
bw/day)
Dosing regime

Body weight 4, (m/f, =-20% at LOAEL at day 42/28, also in recovery 200
groups)
Food consumption 4, at day 5 lactation
200
Food consumption 4, at day 35 and 42
200
Liver
•Abs. liver weight I' (m/f, ,-.-. +45/+29% at LOAEL, also in recovery
40/200
group)
• Rel. liver weight l' (m/f, .. +42/+35% at LOAEL, also in recovery
40
group)
• Centrilobular hepatocellular hypertrophy (m/f, also in recovery
40/200
group)
Immune system
•Thymic atrophy of the cortex (2 animals/24)
•Abs. thymus weight 4, (m, —40% at LOAEL, not in recovery group; f: 200
ns)
• Rel. thymus weight (m/f, ns)
•Abs.+rel. spleen weight (m/f, ns)
• White blood cell count is (m: ns; f, .-+. +35% only in recovery group)
71

1000
1000
1000
200/1000

1000

• kidney, nervous
system, thyroid,
endocrine system

(HirataKoizumi et
al., 2012)

Table 2: Repeated dose toxicity (RDT) animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
NO(A)EL LO(A)EL Key parameters Serum/tissue concentrate- Reference
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
(mg/kg (mg/kg / targets not
ion of PFAS /metabolites
formula
Remarks
bw/d)
addressed
(time of sampling)
to guideline/non-guideline)
bw/d)
All animals: 14 day pre mating

Haematopoietic system:
• Erythrocytes, haemoglobin ↓ (m)
40
200
Males: 42 day
• Reticulocyte ratio ↓ m,↑m: recovery group
200
1000
th
Females: until 5 day of lactation (42• Prothrombine time ↓ (f),
40
200
56 days)
• Basophiles ↑ (f)
200
1000
Clinical chemistry:
• Albumin protein fraction ↑ (m, ≈ +15% at LOAEL, also in recovery
40
200
group; f: only in recovery group)
• α1-globulin protein fraction ↓ (m/f, ≈ –41/–23% at LOAEL, remains 200/40
1000/200
reduced in recovery group)
• α2-globulin fraction (m+f, ns)
• β-globulin protein fraction ↓ (m/f, ≈ –17/–10%, only in recovery
group)
• γ-globulin protein fraction ↑ (m/f, ≈ +68/+59% at LOAEL, not in
200
1000
recovery group)
• AST (m+f, ns)
• ALT ↑ (m, ≈ +53% at LOAEL, f: ns)
200
1000
• ALP ↑ (m/f, ≈ +88/+72% at LOAEL, remains increased in m in
200
1000
recovery group)
• Total bilirubin (m, ≈ +167% at LOAEL, but not in recovery group, f:
200
1000
ns)
• BUN ↑ (m/f, ≈ +41/+41% at LOAEL, not in recovery groups)
200
1000
• Total cholesterol ↓ (f, ≈ –25%, not in recovery group; m: ns)
200
1000
• Triglycerides ↓ (m, ≈ –67% only in recovery group; f: ns)
Other effects
• pancreatic zymogen granules ↓ (m: tendency, f: significant)
200
1000
1000
• Rel. brain weight ↑ (m/f, ≈ +25/+13% at LOAEL, m also in recovery 200
group)
Reproductive and developmental findings:
1000
No significant differences in delivery, live birth and viability indices 200
between control and PFODA treated groups, but a decreasing
tendency in the HD-group.
• body weight of male and female pups ↓
Key parameters/targets
Mortality, body weight, liver, kidney, thyroid, nervous system, immune system, haematopoietic system, endocrine system, clinical chemistry, other effects
Abbreviations/Symbols
/ (forward slash): in the NOAEL and LOAEL columns indicate NOAELs and LOAELs for males/females (unless stated differently); ↑: significant increase; ↓: significant decrease; abs., rel.: absolute, relative; Acox: Acyl
CoA oxidaseALP: alkaline phosphatase; ALT: Alanine aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; CYP4A1: cytochrome P4504A1 or arachidonic acid monooxygenase; C: control; LD:
low dose; MD: medium dose; HD: high dose; m, f: male, female; MoA: mode of action; ns: no significant difference to control; NA: not available, i.e. not determined; T3: triiodothyronine; fT4: free thyroxine; tT4:
total thyroxine; bw: body weight; WT: wildtype; KO: knock out; PPAR- peroxisome proliferator-activated receptor

72

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8. Annex 3: Developmental and reproductive toxicity studies
Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Observed effects
Serum/tissue
maternal
offspring
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

Perfluoroalkyl carboxylic acids (PFCAs)
PFBA: Perfluorobutanoic acid (Syn: Heptafluorobutyric acid, CAS no: 375-22-4, EC no: 206-786-3, Mol. formula: C4HF7O2, MW: 214,xx?; pre-registration process under REACH, self classification available)
17-days
PFBA,
perfluorobuty GD1-GD17
rate,
Mouse (CD1; timed-pregnant)
ammonium
(98%)
Maternal
n/group = 30 (3 blocks)
Offspring sample size?

Maternal

350 mg/kg

• maternal liver weight ↑ (30% est. Fig 2; reversible)

Fertility
• full litter resorptions ↑ (26.9%/350 mg/kg versus 6,8% in control)
• neonatal mortality (no effect)
• number/weight of living foetuses/number of implants (no effect)
Gene expression analysis (neonatal mice): no effects on
PPARa/pregnane X-receptor regulated genes

35

175

175

350

pregnant dams:

(Das et al.,
2008)

2.49 µg/ml serum
0.96 µg/g liver
Pups PND1
0.37 µg/ml serum

0.24 µg/g liver
Developmental
• pups liver weight PND1 ↑ (10-20% est. Fig 5; no longer observed at
35
175
Pups PND10
PND10)
1.12 µg/ml serum
Doses: 0, 35, 175, 350 mg/kg • slightly delayed eye opening (1-1.5 days)
0
35
bw/d
• delayed vaginal opening (2-3 days)
35
175
0.04 µg/g liver
• delayed preputial separation (350/0 mg/kg 33% vs 7%)
175
350
PFHxA: Perfluorohexanoic acid (Syn: Undecafluorohexanoic acid, CAS no: 307-24-4, EC no: 206-196-6, Mol. formula: C6HF11O2, MW: 264,xx?; under PBT assessment under REACH, self classification available)
Exposure: oral (gavage)

PFHxA
ammonium

12-day
GD 6-18 (for both experiments?)

Maternal
• maternal mortality (0: 2/20; 100: 6/20; 350: 1/20; 500: 3/20)

(Iwai and
Hoberman,
2014)

93.4% purity Mouse (CD1; timed-pregnant)

Fertility
• Postnatal mortality (PND 0) ↑ (0:0/217; 100: 0/250; 350: 3/232;
500:21/150)
Maternal:
n/group = 20 (for both experiments?) • Postnatal mortality ↑ (PND 1-4) (0: 2/217; 100: 2/250; 350: 25/229;
500; 20/129)
Exposure: oral (gavage)
• decreased litter size
• neonatal mortality ↑
Two phases i.e. two experiments:
Developmental
Experiment (phase) 1
• weight gain during lactation ↓
Doses: 0, 100, 350, 500 mg/kg bw/d • relative liver weight in F1 ↑

Experiment (phase) 2

Phase 2
increased postnatal mortality (PND 0) (0: 0/249; 7:0/211; 35: 0/232;
175: 4/238)

73

350

500

100

350

350
350

500
500

35

175

Formatted: French (France)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

Doses: 0, 7, 35, 175 mg/kg bw/d

Statistical re-evaluation of Iwai and
Hoberman 2014 confirmed 175 mg/kg
as NOAEL for the reported pup
mortality

PFHxA
(Na-PFHxA)

175

350

comprehensive toxicological evaluation
including

(Loveless
et al.,
2009)

A) One generation reproduction study
Maternal
similar to OECD TG 415
• reduced maternal body weight gain (10-20%)
P0 70d prior to mating until weaning
Developmental
126d (f), 110d (m)
• reduced pup body weight (10-20%)
Rat (SD)

100

500
100

500

100

500

n/sex/group = 20
Exposure: oral (gavage)
Doses: 0, 20, 100, 500 mg/kg bw/d
B) Developmental toxicity study
similarto OECD TG 414
GD6-20/ analysis at GD21
Rat (SD)
n/sex/group = 20

(Iwai et al.,
2019)

Maternal
• reduced maternal body weight gain (19%)
Developmental
• decreased fetal weight (10%)

100

no detectable effects related to reproduction
Exposure: oral (gavage)

74

500

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

Doses: 0, 20, 100, 500 mg/kg bw/d

PFOA: Perfluorooctanoic acid (Syn: Pentadecafluorooctanoic acid, CAS no: 335-67-1, EC no: 206-397-9, Mol. formula: C8HF15O2, MW: 414,xx?; registered under REACH, self classification available)
PFOA

GD1 - birth

ammonium

Mouse (CD1; timed-pregnant)

98% purity
(no information on number per dose)

Maternal
• liver weight ↑ (2-fold < 3 mg/kg)

0
5

• decreased maternal weight gain

For determination of serum
levels:

1
10

rats 10 mg/kg 20 d

• Developmental
• neonatal mortality (Very high in the 20 mg/kg (60%) and 40 mg/kg

3

5

•

199 µg/ml (m)

• growth retardation and and decreased weight among surviving pups

3

5

171 µg/ml (f)

5

10

rats 10 mg/kg 20d

•

Doses: 0, 1, 3, 5, 10, 20,40 mg/kg

mice 20 mg/kg 7 or 17d
mice 20 mg/kg 17 d

(90%) dose groups)
Exposure: oral (gavage)

(Lau et al.,
2006)

• enlarged frontanel, reduced ossification (sternebrae, calvaria), minor

111 µg/ml (m)

• tail and limb defects

0,69 µg/ml (f)
PFOA
ammonium
98% purity

A: pregnant mice (CD-1)
GD1-17
n/group = 10, 12, 11
Exposure: oral (gavage)
Doses: 0, 1, 5 mg/kg bw/d
B: pregnant mice (CD-1)
during gestation (GD1-17/birth)
0 mg/kg (n=7) 1 mg/kg (n=10)
Exposure: oral (gavage)

P0: (only A)
increased neonatal mortality

1

P0: compromised weaning induced mammary involution PND22 (A)
P0: compromised weaning induced mammary involution PND22 (B)
F1: reduced developmental mammary scores (A)
F1: reduced developmental mammary scores (B)

0
0

75

Group only exposed to 5ppb
PFOA (B1)
p0->F2

1
0.00045
0
0

F1 (A&B)
compromised lactational morphology at PND10
(A)
both subgroups of B were also exposed (due to developmental exposure/ treatment of P0 A)
to 5 ppb PFOA in drinking water
(no clear evidence for diminished nutritional support)
(app. 0.00045 mg/kg) starting at GD7 (control groups only exposed to 5ppb in drinking water were
(B)
for the duration of the study
also affected)
F1 (only A)
Offspring generations of B (F1-2)
reduced uterine implants
were continuously dosed with
compromised lactational morphology
5 ppb PFOA (drinking water) until the F2 (A)
end of experiment
Mammary gland scores are consistent with delayed differentiation at

5

0

P0 weaning: 74,8 ng/ml
F1 PND22 21.3 ng/m
F1 PND63 66.33 ng/m
1
F1 dams weaning 86.9 ng/ml
0.00045
F2 pups PND22 26.6 ng/ml
F2 pups PND63 68,5 ng/ml
1
Group P0 1 mg/ml/ 5ppb P0>F2 (B2)

0

0.00045 P0 weaning: 4772.0 ng/ml

1
1

5
5

F1 PND22 2743.8 ng/m
F1 PND63 187.0 ng/m
F1 dams weaning 173.3 ng/ml
F2 pups PND22 28.5 ng/ml
F2 pups PND63 69.2 ng/ml

(White et
al., 2011)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

PND 22 (ns)
group P0 only 5 mg/kg PFOA
(A3)
P0 weaning: 26980.0 ng/ml
F1 PND22 10045 ng/m
F1 PND63 760.3 ng/m
F1 dams weaning 18.7 ng/ml
F2 pups PND22 7.8 ng/ml
F2 pups PND63 1.22 ng/ml
PFOA
90% purity

17-days,
GD1-17
Mouse (ICR; timed-pregnant)

Maternal
• liver weight ↑ (relative weight 5.4-11.4%)

Exposure: oral (gavage)

PFOA

PFOA

5

5
5

10
10

(Yahia et
al., 2010)

•
• Increased GGT, AST, ALP

Maternal sample size:
n/group = 15-19

1

• Reduced globulins, triglycerides and cholesterol

Developmental
• reduced birth weight
• neonatal mortality (100%, 10 mg/kg)

1
1

Doses: 0, 1, 5, 10 mg/kg bw/d
Parental
Two generation study EPA OPPTS
• no reproductive endpoints affected in any generation
8703800
P0 70 day prior to mating (m) or until Developmental (F1)
• birth weights ↓ in relationship with reduced viability
weaning (f)
• Reduced viability not observed in F2
Rat (SD)
• Preputial separation and vaginal opening somewhat delayed
n/sex/group = ? (males and females?)
Parental (F0) doses: 0, 1, 3, 10, 30
mg/kg bw/d
F1: dosing continued after weaning
F2: was maintained through 22d
lactation
Maternal
timed pregnant mice (CD-1)
• abs liver weight ↑ (E11.5) (0: 2.2g; 1: 2.9g, 5: 4.5g)
Litter sample size:
• rel liver weight ↑ (E11.5) (0:5.9%; 1:7.4%, 5: 11.0%)
• enlarged hepatocytes (E17.5)
n/group = 11-13
• abnormal ultrastructure of liver cells (increased
Exposure: oral (gavage)
peroxisomes/mitochondria)
0, 1, or 5 mg/kg bw/d
sacrifice timepoint GD11.5 or GD17.5 •

76

5
5
(Butenhoff
et al.,
2004)

10

0
0
0
0

1
1
1
1

30

(Blake et
al., 2020)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
dosing GD1.5 until 11.5 or GD 1.5 until • Triglycerides (E 11.5) (1 mg/kg 37%, 5 mg/kg 58%)
• Glu ↓ (5 mg/kg E17.5: 20% lower)
17.5
• AST ↑ (5 mg/kg E17.5: up 100%)

PFOA
Ammonium
salt (APFO)
98% purity

Fertility
• embryo weight↓
• placental lesions (nodules, labyrinth congestion or atrophy
Maternal
GD1-17
Mouse (PPARα KO -/- or 129S1/SvlmJ • Maternal liver weight (WT) ↑ (more than 2-fold at 5 mg/kg)
WT), timed-pregnant
• Maternal liver weight (PPARα-KO) ↑ (more than 2-fold at 5 mg/kg)
• Maternal liver weight not dependent on PPARα
Number of litters highly variable
Fertility
n/group = 4-22 litters for individual
• litter loss wt (100%) (1 mg/kg: 70%)
doses
• litter loss wt PPARα KO -/- (100%) (5 mg/kg: 86%)
Exposure: oral (gavage)
• Comparable embryo lethality in WT and PPARα KO mice at 5 mg/kg
Doses: 0.1, 1, 3, 5, 10 or 20 mg/kg
(100% versus 86%)
• Embryo lethality:PPARα-independent
bw/d

0
1
1

1
5
5

1
0

5
1

1
1

5
3

1
10

5
20

(Abbott et
al., 2007)

• neonatal mortality can only be addressed up to 1 mg/kg in WT mice

due to total litter losses at higher exposures
Developmental
• postnatal survival ↓ (WT) (0 mg/kg: 78.9% vs. 1 mg/kg: 42.5% survival
on PND 22)
• postnatal survival ↓ (PPARα-KO) (0 mg/kg: 92% vs. 1 mg/kg > 90%, 3
mg/kg: 87%)

0.3
3

•
• neonatal mortality ↑ (WT: Fig 2A in publication)

0.6
–

• neonatal mortality ↑ (PPARα-KO: Fig 2B in publication)
• neonatal mortality PPARα-dependent
•
• delayed eye opening (WT) (≈ 1 day)

0.3
1

• delayed eye opening (PPARα-KO)

77

Reference

0.6
3

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFOA

Developmental
GD1 - GD17
Male offspring was analyzed at PD 21 • neonatal mortality (PD7)
• decreased number of Leydig cells
and PD70
• altered ultrastructure, markedly increased intracellular substance
Mouse (Kunming)

2.5
1
1

5
2.5
2.5

1

2.5

Reference

(Song et
al., 2018)

•

n/sex/group = 10 (litters or male
pups?)

• Expression of imprinted Dlk-Dio3 gene cluster
•
• no difference in testicular index

Exposure: oral (gavage)

PFOA
ammonium
salt (APFO)
>98% purity

Doses: 0, 1, 2.5 or 5 mg/kg bw/d
Developmental
7-17 days,
GD1-GD17 (full gestation exposure) or • no effects on offspring body weight in both experiments (f+m)
GD10-GD17 (late gestation exposure) • abs/rel liver weight of offspring in full/late gestation exp. (f+m) ↑
Mouse (CD-1), timed-pregnant
• delayed epithelial growth in the mammary gland (full-gestation exp:
PND 63 and 84, on PND 63 no control comparison, but comparison to
Maternal
historical controls…)
• delayed epithelial growth in the mammary gland (late gestation exp:
n/group = 13 (both experiments)
PND1-PND14/PND 14)
Litters
n/group = 13 (equalized to 5 m and 5 f • overall developmental scores ↓ in both experiments (full/late
gestation exp)
in each litter) in full gestation
exposure)
n/group = ca. 11 (15% of dams were
not pregnant (PFOA independent);
litters were mixed and each dam
nursed 7-9 pups with 4-7 females)
Exposure: oral (gavage) (only dams)
Doses (full gestation exposure): 0, 0.3,
1.0, or 3.0 mg/kg bw/d
Doses (late gestation exposure): 0,
0.01, 0.1, or 1.0 mg/kg bw/d
Remark: short acclimation period (only
1 day!): stress responses might
potentiate PFOA effects. On the other
hand, all experiments with purchased
timed-pregnant mice have this
problem

78

(Macon et
al., 2011)
0.3
0.3

0.1
0.3/ 0.01

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFOA
ammonium
salt (APFO)
>98% purity

17 days,
GD1-GD17
Mouse (CD-1 and C57Bl/6); timedpregnant

Developmental
• net body weight (body weight-liver weight) (only CD-1) ↓

1

Reference

(Tucker et
al., 2015)

• no effect on abs liver weight (both strains)
• rel liver weight at PND 21 ↑ (but not at PND35 and 56)(only CD-1

1

mice)
• estradiol and progesterone (ns in both strains)

Maternal sample size:
CD-1 mice in three blocks (block 1 n = •
97, block 2 n = 40, and block 3 n = 26) • no effects on female pubertal events (both strains)
•
C57Bl/6 mice in one block (n = 41)
• mammary gland developmental scores reduced on PND 35 and 56
Litter sample size
(equalized to 10 pups per dam, each 6- (tbc) (CD-1)
• mammary gland developmental scores reduced on PND 21 and 61
7 f + 3-4 m)
(tbc) (C57Bl/6)
Exposure: oral (gavage, in water)

0.01
0.3

Doses: 0, 0.01, 0.1, 0.3 or 1.0 mg/kg
bw/d
Pregnancy rates in CD-1 females were
>60%
the C57Bl/6 block yielded a much
lower rate of approximately 27%
litter with n<5 were excluded
PFNA: Perfluorononanoic acid (Syn: Heptadecafluorononanoic Acid, CAS no: 375-95-1, EC no: 206-801-3, Mol. formula: C9HF17O2, MW: 464,xx?; registered under REACH, self classification available)
PFNA

90-day study,

97% purity

PND25 - PND114 (prepuberty)
Mouse (Parkes strain)
n/sex/group = 7 (m)
Exposure: oral (gavage)
Doses: 0, 0.2, 0.5 mg/kg bw/day

Males
• Body weight (ns)
Fertility
• Abs/rel testis weight (ns)
• spermatozoa motility/viability/number ↓ (≈ –26%/–65%/–37% at
LOEL)
• serum cholesterol ↓ (≈ –33% at LOEL)
• serum testosterone ↓ (≈ –31% at LOEL)
• Litter size ↓ (≈ –42% at LOEL) (reproduction assay)
• testicular proteins/mRNA involved in steroidogenic processes ↓
(StAR/Cyp11A1/3β-HSD/17β-HSD) (≈ –47%/–49%/–35%/–64% at
LOEL)
• Testes lipid peroxidation ↑ (≈ +41% at LOEL)
• Antioxidative enzymes (SOD, CAT) ↓ (≈ –22%/–27% at LOEL)
• number of proliferating testicular cells (PCNA) ↓ (≈ –28% at LOEL)
• Testes apoptosis (Caspase-3 staining) ↑ (≈ +35% at LOEL)

79

(Singh and
Singh,
2019b)
0.2

0.5

0.2
0.2
0.2
0.2

0.5
0.5
0.5
0.5

0.2
–
0.2
0.2

0.5
0.2
0.5
0.5

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFNA

14-day study,

97% purity

PND25 -PND38 (prepuberty)
Mouse (Parkes strain)
n/sex/group = 10 (m)
Exposure: oral (gavage)
Doses: 0, 2, 5 mg/kg bw/day

PFNA
97% purity

14-day study
GD12 – parturition
Mouse (Parkes strain)
Maternal (pregnant females):
n/sex/group = 10
Offspring (male neonates only, killed
on PND3):
n/group = 20 (2 per litter)
n/group = 5 (1 per litter) for testes
sampling
Exposure: oral (gavage) from GD 12

Males
• Body weight gain ↓
Fertility
• intratesticular/serum testosterone ↓ (≈ –20%/–72% at LOEL)
• Rel no. affected seminiferous tubules ↑ (≈ +424% at LOEL)
testicular proteins/mRNA involved in steroidogenic processes (SF1/StAR/Cyp11A1/3β-HSD/17β-HSD) ↓ (≈ –23%/–57%/–50%/–45% /–
46% at LOEL)
• Testes lipid peroxidation ↑ (≈ +74% at LOEL)
• Antioxidative enzymes ↓ (SOD/CAT/GST) (≈ –31%/–35%/–34% at
LOEL)
• number of proliferating testicular cells (PCNA) ↓ (≈ –13% at LOEL)
• Testes apoptosis (Caspase-3 staining) ↑ (≈ +100% at LOEL)
Maternal
• Body weight, birth rate, number of pups per dam, weight of male
pups) (ns)
Developmental
• Abs testis weight of neonatal offspring (ns)
Testis histology (ns)
• Intratesticular level of testosterone in neonatal offspring ↓ (≈ –42 %
at LOEL)
• testicular proteins/mRNA involved in steroidogenic processes (SF1/StAR/Cyp11A1/3β-HSD/17β-HSD) ↓ (≈ –23%/–33%/–37%/–31%/–
25% at LOEL)
• testicular proteins/mRNA involved in gonad development (WT1/SF1)
↓ (≈ –67%/–23% at LOEL)
• Testes PCNA expression ↓ (≈ –32% at LOEL)

2

5

–
–
–

2
2
2

–
–

2
2

–
–

2
2

Reference

(Singh and
Singh,
2019d)

(Singh and
Singh,
2019c)

2

5

2/–/ 2/ 2/ 5/2/5/ 5/
2
5
–/ 2

2/ 5

2

5

Doses: 0, 2, 5 mg/kg bw/day
PFNA

14-day study,

97% purity

PND25 -PND38 (prepuberty)
Mouse (Parkes strain)
n/sex/group = 10 (m)
Exposure: oral (gavage)

Males
• Abs/rel liver weight (≈ +100/42% at LOEL)
–
• Hepatocellular hypertrophy
–
• PPARα expression ↑ (80-fold change at LD, only 5-fold change at HD) –
• Hepatic liver peroxidation ↑ (≈ +26% at LOEL)
–
Fertility
• Testicular glucose ↓ (≈ –22% at LOEL)
–
• Testicular lactate ↓ (≈ –1% at LOEL)
–
• Serum cholesterol ↓ (≈ –19% at LOEL)
2
• Fasting blood glucose ↑ (≈ +16% at LOEL)
–

80

2
2
2
2
2
2
5
2

(Singh and
Singh,
2019a)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

Doses: 0, 2, 5 mg/kg bw/day

PFNA (97%)

Fertility
• Serum testosterone ↓ (≈ –85% at LOEL)
• Serum estradiol ↑ (≈ +104% at LOEL)
• Apoptotic cells in testes ↑ (≈ +367% at LOEL)
n/sex/group = 6 (m)
• Fas gene ex ↑ (≈ +90% at LOEL)
• Bax gene ex ↑ (≈ +36% at LOEL)
• Bcl-2 gene ex ↓ (≈ –27% at LOEL)
Exposure: oral (gavage)
• Vacuolization between Sertoli cells and spermatogonia ↑
• Testicular WT1 protein levels ↑ (≈ +59% at LOEL)
Doses: 0, 1, 3, 5 mg/kg bw/d
• Testicular Transferrin protein levels ↓ (≈ –33% at LOEL)
• Serum MIS ↑ (≈ +22% at LOEL)
Serum inhibin B ↓ (≈ –10% at LOEL)
GD 1-20
Maternal
• Body weight ↓ b(≈ –25%/–11% at GD10/GD21)
Rat (SD)
Fertility
• Birth weight ↓ (≈ –10% for females)
Maternal (pregnant females):
Developmental
n/sex/group = not specified, but since • Systolic blood pressure at 10 weeks of age ↑ (≈ +7/10% for m/f)
• Systolic blood pressure at 26/56 weeks of age (ns)
there were 18-19 litters, there were
• Nephrons per kidney (m) ↓ (≈ –19%)
probably 18-19 dams… (?)
14-day-study
Rat (SD)

PFNA
Purity and
salt not
specified

Offspring:

Remark: Offsping was weaned by non-treated foster-dams! No
n/group = 18-19 litters for birth weight maternal toxicity effects and no lactational effects
n/group = 10-12 pups/litter (litter size Also arsenic trioxide and nicotine bitartrate were also tested as
was standardized)
comparative substances (to PFNA. A second experiment with separate
a control comparing Dex (as positive control; known to increase blood
n/group/sex = 1-2 per litter (not
specified how many litters) for systolic pressure), Atrazine, and PFOS, is also published in this paper.
Body weight gain in PFNA treated animals below those treated with
blood pressure
arsenic. Lowest bw gains were observed with nicotine.
n/group = 5 (1 per litter from 5 litters)
for nephrons (males only)
Exposure: oral (gavage) (only dams)

81

3
3
1
3
3
1
1
–
–
3
–

5
5
3
5
5
3
3
1
1
5
1

(Feng et
al., 2010;
Feng et al.,
2009)

(Rogers et
al., 2014)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

Doses: 0, 5 mg/ kg bw/d

PFNA
>98% purity

28-day RDT study, NTP study, similar to Fertility
OECD TG 407
• Plasma testosterone in m ↓ (≈ –81% at LOEL)
• Plasma testosterone in f ↑ (≈ +30% at LOEL)
Rat (SD)
• L. Cauda Epididymis weight ↓ (m, ≈ –11% at LOAEL)
• L. Epididymis weight ↓ (m, ≈ –7% at LOAEL)
n/sex/group: 10
• Testis weight ↓ (m, ≈ –7% at LOAEL)
• Sperm count (106/g cauda epididymis) ↓ (m, ≈ –18% at LOAEL)
• Germinal epithelium degeneration ↑
Exposure: oral (gavage, in distilled
• Interstitial cell atrophy in testes ↑
water)
• Seminiforous tubule spermatid retention ↑

1.25
−
0.625
−
0.625
0.625
1.25
1.25
1.25

2.5
1.56
1.25
0.625
1.25
1.25
2.5
2.5
2.5

5
1
–
–
–

10
3
1
1
1

5

10

(NTP,
2019b)

Doses: males: 0, 0.625, 1.25, 2.5, 5, 10
mg/kg bw/d; females: 0, 1.56, 3.12,
6.25, 12.5, 25 mg/kg bw/d
PFNA
97% purity

17 days ReproDevTox study (GD1-17), Maternal
non-guideline study
• Maternal body weight at GD13 ↓ (≈ –30% at 10mg/kg bw/d)
• Maternal body weight between GD11-GD17 ↑ (≈ +10% at LOEL)
Mouse (CD-1)
• Abs/rel liver weight (non-pregnant) ↑ (≈ +83%/88% at LOEL)
• Abs/rel liver weight (pregnant) ↑ (≈ +45%/41% at LOEL)
Maternal sample size:
• Abs/rel liver weight (post-weaning) ↑ (≈ +33%/19% at LOEL)
n/group = 1-10 (for pregnancy
Fertility
outcome)
• Pregnancy not carried to term (full litter resorption) at LOEL
n/group = 19-27 (maternal body
• Pregnancy outcome (Implants per litter, abs/rel live foetuses per
weight gain)
litter, fetal weight, skeletal or visceral examinations) (ns)
n/group = 5-15 (liver weights)
Developmental
Offspring sample size:
• Abs/rel fetal liver weight ↑ (≈ +29/22% at LOEL)
n/group = 11-17 litters (postnatal body • Postnatal survival ↓ (≈ 20% survival at LOEL from PND10 onwards)
• Postnatal body weight at PND 7/14/21↓ (≈ –17/24/23% at LOEL;
weight and neonatal survival)
effects persist in male pups until PND 287, but recovers in female
n/group=6-13 litter (offsping body and
pups after PND 49)
liver weight & developmental delay)
• Rel postnatal liver weight at PND10/24↑ (≈ +37/27% at LOEL)

82

Serum concentrations (µg/ml): (Das et al.,
2015)
Pregnant at term:
Control: 0.015 ± 0.003
1 mg/kg bw/d: 15
3 mg/kg bw/d: 25
5 mg/kg bw/d: 80
Not pregnant:
Control: 0.19 ± 0.06
1 mg/kg bw/d: 30
3 mg/kg bw/d: 40
5 mg/kg bw/d: 200
–
3
1

1
5
3

–

1

Post-weaning:
Control: 0.020 ± 0.001
1 mg/kg bw/d: 10
3 mg/kg bw/d: 15
5 mg/kg bw/d: 90

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
• Rel postnatal liver weight at PND 42 ↑ (≈ +18% at LOEL)

Exposure: oral (gavage, vehicle:
deioinized water)
Doses: 0, 1, 3, 5, (10, only till GD13)
mg/kg bw/d

• Delayed development (Eye opening ≈ +13%, preputial separation

≈ +11%, vaginal opening ≈ +10% at LOEL)
• Gene expression heatmapà authors: “PFNA induced a clear PPARαdependent gene expression signature in both fetal and neonatal
mouse liver.” (Rather complex heatmap. PPARα not measured
directly, but only PPARα dependent genes.

Remarks: Increased liver weight of offspring persistent into adulthood.
Remarks: Since overt toxicity was
indicated at 10 mg/kg bw/d, this group Absolute liver weights were significantly increased in the 1, 3 and 5
mg/kg dose groups.
was sacrificed at GD13 and not
However, this effect was not dose dependent, as the 5 mg/kg group
included for further analysis in this
was less affected
study.

1
1

3
3

Liver concentrations (µg/g)
Pregnant at term:
Control: 0.1 ± 0.01
1 mg/kg bw/d: 100
3 mg/kg bw/d: 260
5 mg/kg bw/d: 330
Not pregnant:
Control: 0.67 ± 0.18
1 mg/kg bw/d: 170
3 mg/kg bw/d: 330
5 mg/kg bw/d: 450
After weaning:

“(…) at 43 weeks of age PFNA was still detectable in the liver and serum
(data not shown), indicating the exceptionally slow elimination of this
fluorochemical.”
EFSA Opinion: “The authors also provided benchmark dose estimates.
The most sensitive ones were BMD5/BMDL5 values of 0.43/0.27 mg/kg
bw/day for increased relative liver weights in mothers and
BMD5/BMDL5 of 0.24/0.19 mg/kg bw per day for increased relative liver
weight of pups at PND1.”

Reference

Control: 0.07 ± 0.01
1 mg/kg bw/d: 20
3 mg/kg bw/d: 120
5 mg/kg bw/d: 200
Fetal liver at term (µg/g):
Control: 0.011 ± 0.007
1 mg/kg bw/d: 10
3 mg/kg bw/d: 35
5 mg/kg bw/d: 70

Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)

Serum offspring (µg/ml) (at
PND1àPND10àPND24):

Formatted: Norwegian (Bokmål)

1 mg/kg bw/d: ≈20à10à1
3 mg/kg bw/d: ≈50à20à5
5 mg/kg bw/d: ≈75à80à25

Formatted: Norwegian (Bokmål)

Liver offspring (µg/g) (at
PND1àPND10àPND24àPND42
àPND70):
1 mg/kg bw/d:
≈55à60à20à10à5
3 mg/kg bw/d:
≈150à150à55à30à20

83

Formatted: Norwegian (Bokmål)

Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)
Formatted: Norwegian (Bokmål)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

5 mg/kg bw/d:
≈205à210à175à35à40

PFNA (97%)

GD 1-PND21 (42-days for nonpregnant Maternal
females)
• Body weight (ns)
–
0.83
Mouse (129S1/SvlmJ WT and PPARα • Abs/rel liver weight (non-pregnant WT) ↑ (≈ +73/72% at LOEL)
0.83/– 1.1/0.83
KO on 129S1/SvlmJ background (Ppara-• Abs/rel liver weight (pregnant WT) ↑ (≈ +26/12% at LOEL)
• Abs/rel liver weight (non-pregnant KO) ↑ (≈ +46/26% at LOEL)
1.1/0.83 1.5/1.1
tm1Gonz/J))
Fertility
• Implantation and total litter size (ns)
n/sex/group = 9-18 pregnant females, • Number of live pups at birth (WT) ↓ ( at LOEL)
1.1
8-16 litters
• Full litter resorption or whole litter loss (WT) ↑ (35% litter loss at
0.83
2
LOEL)
1.5
• Pregnancy rate (KO) ↓
Exposure: oral (gavage)
Developmental
• Survival of pups (PND0-PND21) (WT) ↓ (≈ –64% at LOEL)
0.83
Doses: 0.83, 1.1, 1.5, 2 mg/kg bw/d
• Eye opening ↑ (i.e. delay) (WT) (+2 days at LOEL)
1.5
• Pup birth weight (ns)
• Pup weight gain (PND7-post-weaning) (WT f) ↓ (≈ –25% at LOEL)
1.5
• Pup body weight (PND21, WT, sexes combined) ↓ (≈ –21% at LOEL)
1.5
• Pup abs/rel liver weight (PND 21, WT, sexes combined) ↑ (≈ +45/42%
–
at LOEL)
• Pup rel liver weight (PND 21, KO, sexes combined) ↑ (≈ +22% at LOEL)
1.5
Remarks: non pregnant = mice that had a vaginal plug, but had full
litter resorption
KO mice with no live pups take up more PFNA than WT but show
less/weaker effects
KO mice with live pups have less PFNA in serum than WT
KO pups show higher PFNA serum levels compared to WT (Pregnant KO
mice transfer more PFNA to offspring?)
Mice with live pups showhigher variance in serum PFAN than mice with
no live pups (WT and KO)

Serum PFNA (µg/ml) in
females with no live pups
(WT/KO):

1.1
2
2
2
0.83
2

Control: 0.067 ± 0.005/
0.048 ± 0.008
0.83 mg/kg bw/d: 28.5 ± 1.22/
38.4 ± 2.34
1.1 mg/kg bw/d: 39.7 ± 1.26/
53.9 ± 2.51
1.5 mg/kg bw/d: 48.4 ± 1.54/
72.1 ± 2.91
2 mg/kg bw/d: 64.0 ± 2.46/
83.4 ± 2.93
Serum PFNA (µg/ml) in
females with live pups
(WT/KO):
Control: 0.022 ± 0.004/
0.016 ± 0.001
0.83 mg/kg bw/d: 8.91 ± 1.51/
2.76 ± 0.172
1.1 mg/kg bw/d: 23.2 ± 2.57/
4.17 ± 0.310
1.5 mg/kg bw/d: 21.0 ± 3.01/
11.8 ± 5.71
2 mg/kg bw/d: 35.3 ± 3.90/
22.6 ± 5.69
Serum PFNA (µg/ml) in pups
(WT/KO):
Control: 0.033 ± 0.008/
0.068 ± 0.027
0.83 mg/kg bw/d: 9.60 ± 9.37/
15.2 ± 1.01

84

Formatted: Norwegian (Bokmål)

(Wolf et
al., 2010)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

1.1 mg/kg bw/d: 15.7 ± 1.42/
19.4 ± 0.69
1.5 mg/kg bw/d: 17.5 ± 1.15/
26.4 ± 1.39
2 mg/kg bw/d: 25.3 ± 2.70/
38.4 ± 1.80
S-111-S-WB 2-generation (>70 days before mating Mortality (1 F0 male died in study week 14, “moribund condition of
(mixture,
until weaning), OECD TG 416 (for repro this animal was attributed to test article administration)
main
Parental (F0+F1)
tox)
component Rat (SD)
• Body weight (F0, m) (study weeks 0-7) (ns)
PFNA) (15.2%
• Body weight (F0, m) (study week 7-18) ↓ (≈ –25% after study week 18
in water,
at LOEL)
doses already n/sex/group = 30 (F0 and F1
• Food consumption (m, pre-mating weeks 0-10) ↓ (data not shown)
generation)
corrected)
• Abs/rel liver weight (F0, m) ↑ (≈ +22/22% at LOEL)
• Abs/rel liver weight (F0, f) ↑ (≈ +16/14% at LOEL)
CAS No. of
• Abs/rel liver weight (F1, m) ↑ (≈ +16/17% at LOEL)
mixture:
Exposure: Oral (gavage)
• Abs/rel liver weight (F1, f) ↑ (≈ +16/14% at LOEL)
72968-38-8
• Abs/rel kidney weight (F0, m) ↑ (≈ +9/9% at LOEL)
Doses: 0, 0.025, 0.125 and 0.6 mg/kg • Abs/rel kidney weight (F0, f) ↑ (≈ +7/7% at LOEL)
bw/day
• Abs/rel kidney weight (F1, m) ↑ (≈ +10/11% at LOEL)

0.6

Serum concentrations
Stump et
analysed for C8 (PFOA9, C9
al. 2008
(PFNA), C11 (PFUnDA) and C13
(PFTrDA) (ng/ml):

0.6

Cmax (peak 0-16 h)
F0 females (Day 64):

0.125
0.6
0.125
0.6
0.125
0.125
0.125

•
• Hepatocellular hypertrophy (F0, m) ↑ (mild/moderate)
•
• Hepatocellular hypertrophy (F0, m) ↑ (mild/moderate)
• Hepatocellular necrosis (F0, m) ↑ (minimal/mild)
•
• Hepatocellular centrilobular vacuolation (minimal and mild)
• Renal tubule cell hypertrophy (F0, m) ↑ (minimal/mild)
• Renal tubule cell hypertrophy (F0, f) ↑ (minimal and mild)

Fertility (F0+F1)
• No effects on fertility indices observed (F0+F1, m+f)
• Sperm motility/progressive motility (F0, m) ↓ (≈ –5/6% at LOEL)
• Epididymis weight (F0/F1, m) ↓ (≈ –12/10% at LOEL)
• Epididymal sperm concentration (F0, m)↓ (≈ –13% at LOEL)
• No effects on spermatogenic endpoints (testicular sperm numbers
and sperm production rate, and morphology)
Developmental (F1+F2)
• No effects on pups (number of litters,, litter size, pup weight, vaginal
patency/balanopreputial separation)

85

0.025/0.
125
0.6
0.025/0.
125
0.025
0.125/0.
6
0.6

Control: ≈ 100 (?)
0.125 mg/kg bw/d: ≈ 3000
0.6 mg/kg bw/d: ≈ 16000
F0 females (GD19):
Control: ≈ 100 (?)
0.125 mg/kg bw/d: ≈ 2500
0.6 mg/kg bw/d: ≈ 1400
Mean concentration on LD13
at 2 h post-dosing:
Dams:
Control: ≈ 500 (?)
0.125 mg/kg bw/d: ≈ 1500
0.6 mg/kg bw/d: ≈ 7500
Pups (m+f similar)
Control: ≈ 500 (?)

0.6
0.6
0.6

0.125 mg/kg bw/d: ≈ 2000
0.6 mg/kg bw/d: ≈ 10000

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

• Body weight (F1, m) (post-weaning week 1-10) (ns)
• Body weight (F1, m) (post-weaning week 11-22) ↓ (≈–32% in post-

0.6

weaning week 22)
• Body weight (F1+F2, m+f) (final (ns)
• Rel liver weight of pups (F1, m/f) ↑ (≈ +7/8% at LOEL)
• Rel liver weight of pups (F2, m/f) ↑ (≈ +13/11% at LOEL)

0.125
0.6

•
• Subacute liver inflammation (F1, m) ↑ (minimal)
• Hepatocellular hypertrophy (F1, m) ↑ (minimal and mild/moderate)
•
• Minimal lymphocyte infiltrate (F1, m)
• Hepatocellular necrosis (F1, m) ↑ (minimal)
• Renal tubule cell hypertrophy (F1, m) ↑ (mild)

0.025
0.025/0.
125
0.6
0.025
0.6

Remark: additional 8 females of F0 for toxicokinetics from > 70 days
prior to mating till GD19
Weaning on lactation day (LD) 21, afterwards selected F1 animals
were continued on oral gavage (same doses as parents)
Litters were reduced to 10 pups per litter (5 f, 5 m, if possible) on PND
4, on PND 13 8 per litter (4 f, 4 m if possible)
Food efficiency was statistically significantly lower. Reduced body
weight gain is not (only) a result of reduced food uptake.

In summary, the most sensitive endpoint after gestational exposure to PFNA was increased liver weight in both maternal and offspring CD-1 mice, and a reduction in postnatal weight gain in F1, EFSA 2020
with an LOAEL of 1 mg/kg bw per day, with corresponding concentration in serum from the dams at term of 20 lg/mL. Delay in development was seen at 3 mg/kg bw per day, and at 5 mg/kg bw (summary
per day, there was an increase in neonatal mortality. A 90-day male reproductive study reported decreased sperm production, decrease in cholesterol, steroidogenic enzymes and testosterone, as at the end
well as decreased number of pups in the next generation, with an NOAEL and LOAEL of 0.2 and 0.5 mg/kg bw per day, respectively. Effects on male reproduction parameters were also reported by of Repro
NTP for rats at somewhat higher exposure levels, and it was noted that 28 days is shorter than one spermatogenic cycle and too short to fully assess male reproductive parameters.
effects of
PFNA)
PFDA: Perfluorodecanoic acid (Syn: Nonadecafluorodecanoic acid, CAS no: 335-76-2, EC no: 206-400-3, Mol. formula: C10HF19O2, MW: 514,xx?; registered under REACH, self classification available)

86

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFDA
96%

4/10-day DevTox Study, non-guideline Maternal (GD 10-13):
• maternal body weight gain ↓ (≈ –61% at LOEL)
Mouse (C57BL/6N)
• Abs/rel liver weight ↑ (≈ +10/3% at LOEL)
Maternal (GD 6-15):
n/group (maternal) = 10-13/10-14
• Maternal body weight gain ↓ (≈ –92% at LOEL)
• Abs/rel liver weight ↑ (≈ +61%/+127% at LOEL)
n/group (litters) = 10-13/7-14
n/group (foetuses) = 83-106/23-53
Exposure: oral (gavage, in corn oil)
Doses: 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32
mg/kg bw/day on gestation days (GD)
10-13 (4 consecutive days)

(Harris and
Birnbaum,
1989)

8
16
0.5/0.25 1/0.5
3
0.3

6.4
1

Fertility (GD 10-13):
• Live foetuses per litter ↓ (≈ –32% at LOEL, but ns)
• Resorptions per litter ↑ (ns) (≈ +170% at LOEL)
Fertility (GD 6-15):
• Live foetuses per litter ↓ (≈ –46% at LOEL)
• Resorptions per litter ↑ (≈ +344% at LOEL, but p=0.06)

Developmental (GD 10-13):
• Fetal body weight ↓ (≈ –10% at LOEL)
Developmental (GD 6-15):
• Fetal body weight ↓ (≈ –3% at LOEL)
• Absence of fifth sternabrae (15% of examined foetuses at LOEL)
• Delay in braincase ossification (overall) (26% of examined foetuses at
LOEL)
Delay in phalanges ossification (18% of examined foetuses at LOEL)
PFDA (>98%) 28-day RDT study, NTP study, similar to Fertility
0.625
• Plasma testosterone in m ↓ (≈ –64% at LOEL)
OECD TG 407
• Plasma testosterone in f ↑ (≈ +32-355% at LOEL)
0.156
Rat (SD)
• L. Cauda Epididymis weight ↓ (m, ≈ –11% at LOAEL)
0.625
• L. Epididymis weight ↓ (m, ≈ –10% at LOAEL)
0.625
n/sex/group = 10
• Testis weight ↓ (m, ≈ –11% at LOAEL)
1.25
• Sperm number?(106/g cauda epididymis) ↓ (m, ≈ –30% at LOAEL)
1.25
0.625
Interstitial cell atrophy in testes ↑ (m, 80% incidence at LOAEL)
Exposure: oral (gavage, in distilled

Reference

or 0, 0.03, 0.1, 0.3, 1, 3, 6.4, or 12.8
mg/kg bw/day, on GD 6-15 (10
consecutive days)

1.25
0.312
1.25
1.25
2.5
2.5
1.25

16
16

32
32

6.4
6.4

12.8
12.8

0.25

0.5

0.03
3.0
–

0.1
6.4
0.03

3.0

6.4
(NTP,
2019b)

water)
Doses: males: 0, 0.156, 0.312, 0.625,
1.25, or 2.5 mg/kg g bw/d
PFUnDA: Perfluoroundecanoic acid (Syn: Henicosafluoroundecanoic acid, CAS no: 2058-94-8, EC no: 218-165-4, Mol. formula: C11HF21O2, MW: 564,xx?; SVHC, candidate for authorisation under REACH, self
classification available)

87

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFUnDA
(98.5%)

42-day combined RDT with the
Repro/DevTox Screening Test (OECD
TG 422)
14 days before mating until day 4 of
lactation
Rat Crl:CD (SD)
n/sex/group = 12 (+5 of highest dose
and control for 14-day recovery)
Exposure: oral (gavage, vehicle: corn
oil)
Doses: 0, 0.1, 0.3, 1.0 mg/kg bw/d

Parental (m/f)
• Grip strength of forefoot ↓ (no data shown)
0.3
• Body weight (female satellite group) ↓ (day 38-41+ 14-day recovery; –
similar in males, but ns)
• abs. liver weight ↑
0.3/0.3
• rel. liver weight ↑
0.1/0.3
• Hepatocyte hypertrophy ↑ (≈ 58-52% incidence at LOAEL)
0.3/0.3
• rel./abs. spleen weight ↓ (m only, ≈ −19-23% at LOAEL)
0.3/0.3
• serum ALP, ALT ↑ (m only, ≈ +26% for ALT and +139% for ALP)
0.3
• serum urea nitrogen ↑ (≈ +46-61% at LOAEL)
0.3/0.3
• serum albumin ↓ (m only, ≈ −7% at LOAEL)
0.3
• total protein ↓ (≈ −10-11% at LOAEL)
0.3/0.3

1
1

Reference

(Takahashi
et al.,
2014)

1/1
0.3/1
1/1
1/1
1
1/1
1
1/1

Fertility (m/f)
• No effects on epididymis, testis/uterus, estrous cycle or
delivery/nursing (ns)
Developmental
• No abnormal findings in general appearance of pups
• Body weights (PND0) (m/f) ↓ (≈ –13/12% at LOEL)
• Body weights (PND4) (m/f) ↓ (≈ –19/16% at LOEL)

0.3
0.3

1
1

PFDoDA: Perfluorododecanoic acid (Syn: Tricosafluorododecanoic acid, CAS no: 307-55-1, EC no: 206-203-2, Mol. formula: C12HF23O2, MW: 614.10; SVHC substance, candidate list for authorisation, self
class.available)
PFDoDA
(97/%)

42-day RDT study, OECD TG 422, 14
day recovery (0, 2.5 mg/kg bw/d)
Rat (SD)
n/sex/group = 12

Maternal
• mortality of pregnant females (7/12 dead)

0.5

2.5

0.5
0.5
0.5

2.5
2.5
2.5

0.2

0.5

(Kato et al.,
2015)

Fertility
• abs. L. Epididymis weight ↓ (m, ≈ −32% at LOAEL)
• spermatid and spermatozoa counts (m) ↓
• discontinuous dioestrus (f)

Exposure: oral (gavage)
Doses: 0, 0.1, 0.5, 2.5 mg/kg bw/d (14day recovery group: 0, 2.5 mg/kg
bw/d)
PFDoDA

110 day RDT study

Paternal
• Body weight ↓ (≈ −5.5% at LOEL)

88

(Shi et
al.,

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
95%

Rat (SD)

Reference
2009)

Fertility
• Abs/rel weight of testis, prostate, seminal vesicle and vas deferens

n/sex/group = 6 (m)

(ns)
• Serum testosterone ↓ (≈ −44% at LOEL)

0.05

0.2

0.2
-

0.5
0.02

0.02
0.05

0.05
0.2

0.2
Testicular gene expression of LH ↓ (≈ – 50%, only at 0.05mg/kg bw/d)
Maternal
• Body weight ↓ (-6% at LOEL)
1.5
Fertility
• Serum total cholesterol ↑ (≈+32% at LOEL)
1.5
• Serum estradiol ↓ (≈–37% at LOEL)
1.5
• Serum FSH+LH (ns)
• Ovarian gene expression ↓ (≈–35% for LHR, ≈–40% for StAR, ≈–36%
1.5
for P450SCC, ≈–36% for ER-β at LOEL)
• Ovarian gene expression ER-α ↓ (≈–36% at LOEL)
0.5
• Ovarian gene expression 17β-HSD ↑ (≈+110% at LOEL)
–
Remark: “P450SCC is a rate limiting enzyme responsible for the
conversion of cholesterol to pregnenolone in estrogen biosynthesis.”

0.5

• Serum LH, FSH, cholesterol (ns)

Exposure: oral (vehicle: Tween-20)

• Cast-off cells in some seminiferous tubules in testes (no quant)
• Testicular gene expression of StAR ↓ (≈ −35% at LOEL)

• Testicular gene expression of P450scc ↓ (only at 0.05)
Doses: 0.02, 0.05, 0.2, 0.5 mg/kg bw/d • Testicular gene expression of IGF-I ↓ (≈ −25% at LOEL)
• Testicular gene expression of IGF-IR and IL-1α ↓ (≈ −25%/–30% at

LOEL)
• Testicular gene expression of GnRH-R and FSH ↓ (≈ −45% at LOEL)

PFDoDA

28 day RDT study

95%

PND24 till PND52-55 (i.e 28-31-days,
sample collection in dietrous stage,
pubertal females)
Rat (SD)
n/sex/group = 8 (f)
Exposure: oral (vehicle: Tween-20)

3

(Shi et
al.,
2009)

3
3
3
1.5
0.5

Doses: 0, 0.5, 1.5, 3 mg/kg bw/d
PFDoDA

14-days RDT study

95%

Rat (SD)

Males
• Body weight ↓ (≈ −25.2% at LOEL)

5

5
5
1

10
10
5

5

10

5
1
1
1
1

10
5
5
5
5

Fertility
• Total serum cholesterol ↑ (≈ +38.5% at LOEL)

n/sex/group = 6 (m)

1

• Serum LH ↓ (≈ −30% at LOEL)(FSH no effect)

• Testosterone ↓ (≈ −50% at LOEL)
Exposure: oral (gavage, vehicle: Tween- • (Estradiol ↓ (only at MD, ≈ –50%))
• Apoptotic features present in Leydig cells, Sertoli cells and
20)
spermatogenic cells
• Abs testis weight ↓ (≈ −22.4% at LOEL)
Doses: 1, 5, 10 mg /kg bw/d
• Rel testis weight ↑ (≈ +36% at LOEL)
• Testicular gene expression of SR-B1 ↓ (≈ −60% at LOEL)
• Testicular gene expression of StAR ↓ (≈ −50% at LOEL)
Testicular gene expression of P450scc ↓ (≈ −90% at LOEL)

89

(Shi et al.,
2007)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFDoDA

14-day RDT study
Rat (SD)

Males
• Body weight ↓ (≈ −21% at LOEL)

5

Reference

(Chen et
al., 2019)

10

Fertility
• Abs. testis weight ↓ (≈ –8% at LOEL)

5
10
–
5
• Serum LH ↓ (≈ –36% at LOEL)
–
5
• Serum FSH ↓ (≈ –9% at LOEL)
–
5
Exposure: oral (gavage)
• Gene expression of Leydig cell genes from testis ↓ (≈ –29% for Lhcgr, –
5
≈–47% for Cyp11a1 at LOEL)
Doses: 0, 5, 10mg/kg bw/d
• Gene expression of Leydig cell genes from testis ↓ (≈ –57% for
5
10
Scarb1, ≈–59% for Star, ≈ –61% for Cyp17a1, ≈ –53% for Hsd11b1 at
LOEL)
• Gene expression in sertoli cells (ns)
• Protein levels of Leydig cells (similar to gene expression)
• Sirt1 signalling ↓ (≈ –33% at LOEL)
–
5
5
PGC-1α signalling ↓ (≈ –35% at LOEL)
10
In summary, exposure of rats to PFDoDA prior to and during gestation induced maternal and reproductive effects (continuous dioestrus and fetal loss) with an NOAEL of 0.5 mg/kg bw per day.
Male reproductive effects (decreased spermatid and spermatozoa counts) were seen at a similar NOAEL of 0.5 mg/kg bw per day, which is higher than the NOAEL of 0.1 mg/kg per day observed
for repeated dose toxicity in the same experiment.
n/sex/group = 8 (m)

• Serum testosterone ↓ (≈ –82% at LOEL)

EFSA
(summary
at the end
of Repro
effects of
PFDoDA)

PFTeDA: Perfluorotetradecanoic acid (Syn: Heptacosafluorotetradecanoic acid, CAS no: 376-06-7, EC no: 206-803-4, Mol. formula: C14HF27O2, MW: 714.11; SVHC, candidate list for authorisation; Self class.
available, but only Skin Corr. 1B)
PFTeDA,
Repeated dose and
Perfluorotetra reproductive/developmental toxicity
decanoic acid (OECD TG 422)

n/sex/group = 12

Parental
• Males: ↓ body weights on day 7 and day 14 (treated animals versus
recovery group)
• Females: ↓ body weight on day 4 of lactation
• Females: ↓ body weight during lactation period
• Males: ↑ absolute and relative liver weight
• Females: ↑ relative liver weight

Exposure: oral (gavage, vehicle 0.5%
carboxymethylcellulose sodium in
water)

Fertility
• No effects on estrous cycle, copulation index, fertility index, gestation
length, Corpora lutea, implantation sites, delivered pubs, sex ratio

Doses: 0, 1, 3, 10 mg/kg bw/day)

Developmental effects
• Body weight of pups (m/f) ↓ (≈–14/13% on PND 1, ≈–18/17% on
PND4 at LOEL)

Rat (SD)

Dosing regime

90

3

10

1
3
1
3

3
10
3
10

(HirataKoizumi et
al., 2015)

3

10

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)

Reference

All animals: 14 day pre-mating
Males: 42 day
Females: during lactation up to 5 days
after parturation (app. 42 days i.e. 14
premating, 1-2 days mating 21-23
gestation, plus 5 days)
High dose recovery groups (14 days
recovery after treatment)
PFHxDA: Perfluorohexadecanoic acid (Syn: Perfluoropalmitic acid, CAS no: 67905-19-5, EC no: 267-638-1, Mol. formula: C16HF31O2, MW: 814.13; only self class. Skin Corr. 1B , no further ECHA data)
Repeated dose and
PFHxDA,
Perfluorohexa reproductive/developmental toxicity
decanoic acid (OECD TG 422)
Rat (SD)
n/sex/group = 12
Exposure: oral (gavage, vehicle 0.5%
carboxymethylcellulose sodium in
water)

Parental
• Males: ↓ body weights on day 35 and day 42 of administration

20

100

20
20

100
100

(HirataKoizumi et
al., 2015)

period
• Males: ↓ food consumption decreased at day 14 of recovery period
• Females: ↓ food consumption GD 5 - 10 gestation; LD 4

Developmental
• Body weight of offsping (m/f) ↓ (≈–4/5% on PND1, ≈–6/6% on PND4
at 100 mg/kg bw/d, but not significant)
• no other effects are reported

Doses: 0, 4, 20, 100 mg/kg bw/day)
Dosing regime:
All animals: 14 day pre mating
Males: 42 day
Females: during lactation up to 5 days
after parturition
High dose recovery groups (14 days
recovery after treatment)
PFODA: Perfluorooctadecanoic acid (Syn: Perfluorostearic acid, CAS no: 16517-11-6, EC no: 240-582-5, Mol. formula: C18HF35O2, MW: 914.15;

91

)

Table 3: Developmental and reproductive toxicity animal studies (abbreviations, symbols and key parameters/targets explained underneath the table)
Substance study design, species, route of
Serum/tissue
maternal
offspring
Observed effects
(%), EC/CAS, exposure, doses (guideline/similar
NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS
formula
Remarks
/metabolites
to guideline/non-guideline)
PFODA,
Repeated dose and
Perfluoroocta reproductive/developmental toxicity
decanoic acid (OECD TG 422)
Rat (SD)

Parental
• Females: ↓ body weight after 14 days (~20% at day 28) ↓ lower food 200
consumption at day 5 lactation
200
• Males: ↓body weight after 28 days (~20% at day 42)
200
• abs. and rel.liver weight (f+m) ↑ (≈ +66% abs. in HD)
40

1000
1000
1000
200

n/sex/group = 12

Fertility
• number of corpora lutea ↓ (≈–16% at LOEL)
Exposure: oral (gavage, vehicle 0.5% • number of implantations ↓ (≈–15% at LOEL)
• Total number of pups born ↓ (≈–28% at LOEL)
carboxymethylcellulose sodium in
• No of live pups on PND0/PND4↓ (≈–35/5% at LOEL)
water)
• No significant differences in delivery index and viability indices
between control and PFODA treated groups, but a decreasing
Doses: 0, 40, 200, 1000 mg/kg bw/day tendency in the HD-group.
Dosing regime:
Developmental
All animals: 14 day pre mating
• Body weight pups (m/f) ↓ (=–23/23% on PND0, ≈–27/26% on PND1,
Males: 42 day
≈–-28/26% on PND 4 at LOEL)
Females: until 5th day of lactation (42- Body weight gain (m/f) ↓ (≈–37/32% at LOEL)
56 days)

92

200
200
200
200

1000
1000
1000
1000

200

1000

200

1000

200

1000

Reference

(HirataKoizumi et
al., 2012)

9. Annex 4: Carcinogenicity studies
Table 4: Animal studies in relation to carcinogenic effects (abbreviations, symbols and key parameters/targets explained underneath the table)
PFCA (%) study design
Observed effects
PFHxA

104-week RDT carcinogenicity study
Rat (SD)

NO(A)EL LO(A)EL Reference

no indication for neoplasia/hyperplasia

(Klaunig et
al., 2015)

n/sex/group = 60 (except HD with 70)
Exposure: oral (gavage)
Doses: 0, 2.5, 15, 100 (m); 0, 5, 30, 200 (f)
mg/kg bw/d

PFOA

Neoplasia/hyperplasia
Liver
• Liver adenoma: 6%, 2%, and 10% in males and 0%, 0%, and 2% in females in C, LD, HD
Reproductive tissue
n/sex/group = 50-65
1.5/–
• Leydig cell adenoma ↑ (0, 4, 14% in C, LD, HD)
–
• mammary gland adenoma in females: 15%, 31%, and 11% in C, LD, HD
Exposure: oral (dietary, ad libitum)
1.5
• fibroadenomas in mammary glands of females: 22%, 42%, and 48% in C, LD, HD
Doses: 0, 30, 300 ppm; corresponding to Pancreas
approximately 0, 1.5, 15 mg/kg bw/d (0, • pancreatic acinar cell hyperplasia: 0%, 4%, 4% in C, LD, HD from original study; and after re-evaluation of male tissue
1.5/–
1.3 or 14.2 mg/kg bw/d for males and 0, samples by Caverly-Rae et al. (2014): 6.5%, 2%, and 21% in C, LD, HD with significant increase in HD
Caverly-Rae et al. (2014): “A pathology peer review of male exocrine pancreatic tissues from the earlier study,
1.6 or 16.1 mg/kg bw/d for females)
conducted in 1981–1983 by Butenhoff et al., was undertaken. This review identified an increase in acinar cell
hyperplasia but not adenoma or carcinoma in the earlier study.”

(3M, 1983)

24-months RDT study
Rat (SD),

PFOA (98- 24-months RDT study
100%)
Rat (Crl:CD BR (CD))

(Sibinski,
1987)
15/–
–
15

15/–

93

(Hardisty et
al., 2010)
(Caverly Rae
et al., 2014)

The only statistically significant neoplastic lesions were an increase in testicular Leydig cell
adenomas and fibroadenomas in mammary glands (Butenhoff et al., 2012b); a re-evaluation of the original slides by a
Pathology Working Group resulted in no significant increase of fibroadenomas with following incidences: 36, 44, 46%
in C, LD, HD (Hardisty et al., 2010)
Neoplasia/hyperplasia

Liver
• Liver adenoma ↑ (+1200% (13% vs. 1% in C))
n/sex/group = 156 (males only)
n/group (neoplasia/hyperplasia) = 76-80 Pancreas
• Acinar cell hyperplasia ↑ (+290% (39% vs. 10% in C))
• Acinar cell adenoma ↑ (+800% (9% vs. 1% in C))
Exposure: oral (dietary)
Reproductive tissue
• Leydig cell hyperplasia ↑ (+39% (46% vs. 33% in C))
Doses: 0, 300 ppm (equivalent to 13.6
• Leydig cell adenoma ↑ (+267% (11% vs. 3% in C))
mg/kg bw/d)

(Butenhoff
et al.,
2012b)

(Biegel et
al., 2001)
–

13.6

–
–

13.6
13.6

–
–

13.6
13.6

Table 4: Animal studies in relation to carcinogenic effects (abbreviations, symbols and key parameters/targets explained underneath the table)
PFCA (%) study design
Observed effects

NO(A)EL LO(A)EL Reference

107-weeks RDT study with perinatal and Neoplasia/hyperplasia
Non-neoplastic lesions were only observed in the liver and pancreas of male rats
post-weaning exposure
Rat (SD)
Liver
• Hepatocellular adenoma and carcinoma in males of 0/40, 300/40, 0/80, and 300/80 ppm groups ↑ (0/0, 0/20, 0/40,
n/sex/group:
1.1
0/80: 0%, 0%, 14%, 22%; 300/0, 300/20, 300/40, 300/80: 0%, 2%, 10%, 24%)
study 1: F0 females: 103 (C) or 36 (150
and 300 ppm groups); F1 rats: 60 m and • higher incidences of hepatocellular carcinomas in the 300/80 ppm group compared to the 0/80 group
60 f; study 2: F0 females: 147, F1 males:
Pancreas
60 (no females)
• acinar cell adenomas and adenocarcinomas ↑ in males of all groups exposed post-weaning (0/0, 0/20, 0/40, 0/80:
6%, 58%, 52%, 64%; 300/0, 300/20, 300/40, 300/80: 14%, 40%, 60%, 60%)
Exposure: oral (diet)
• occurrence of acinar cell adenomas and adenocarcinomas in the 0/1,000 and 300/1,000 ppm female groups (not
statistically significant)
Doses:
study1: perinatal: 0, 150, 300 ppm; postweaning: m: 0, 150, 300 ppm (≈ 0, 16, 32 clear evidence of carcinogenic activity of PFOA in male SD rats based on the increased incidence of hepatocellular
neoplasms (predominately hepatocellular adenomas) and increased incidence of acinar cell neoplasms
mg/kg bw/d), f: 0, 300, 1000 ppm (≈ 0,
(predominately acinar cell adenomas) of the pancreas; there was no increase in the incidence of Leydig cell
30, 100 mg/kg bw/d)
neoplasms observed in this study, possibly due to lower doses compared to two previous studies (3M, 1983; Biegel et
study 2: perinatal: 0, 300 ppm; post
weaning: 0, 20, 40, 80 ppm (≈ 0, 1.1, 2.2, al., 2001)
4.6 mg/kg bw/d)
PFOA
52-week RDT study to assess tumorNeoplasia/hyperplasia
(purest
promoting activity of PFOA
available Rat (Wistar, 180 g)
Liver
• Hepatocellular carcinoma of Group B (C: 0/7, 0.005%: 1/7, 0.02% PFOA: 5/9) and in triphasic protocol ↑ (C: 0/7;
analytical
0.015%: 4/12)
grade)
n/sex/group = 15 (m)
• No liver tumours occurred in the non-initiated rats receiving PFOA
• PFOA also selectively induced the peroxisomal acyl-CoA oxidase activity and, to a lesser extent, catalase activity
Exposure: oral (diet, ad libitum)

(NTP,
2019a)

PFOA
(>98%)

2.2

1.1

(Abdellatif
et al., 1991)

Doses: 0, 0.005, 0.02% PFOA (Group A, B)
or 0.015% (triphasic protocol) in diet
initiation: Group A – no initiation; Group
B - 200 mg/kg diethylnitrosamine;
PFOA increased the incidence of malignant hepatocellular carcinoma and also selectively induced the peroxisomal
triphasic protocol - 200 mg/kg
diethylnitrosamine + 0.03% 2-AAF in diet acyl-CoA oxidase activity and catalase activity
for 2 weeks + single oral dose of CCl4 (2
mL/kg)
positive control: phenobarbital (0.05%)
PFNA,
PFOS,
PFOA,

6 months RDT study
trout
A two-stage chemical carcinogenesis
model in trout to evaluate PFOA, PFNA,

Neoplasia/hyperplasia

(Benninghof
f et al.,
2012)

94

Table 4: Animal studies in relation to carcinogenic effects (abbreviations, symbols and key parameters/targets explained underneath the table)
PFCA (%) study design
Observed effects
PFDA or
8:2FTOH

PFOA,
PFOS

PFDA, PFOS, and 8:2 fluorotelomer
alcohol (8:2FtOH) as complete
carcinogens or promoters of aflatoxin B1
(AFB1)- and/or
N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG)-induced liver cancer.

NO(A)EL LO(A)EL Reference

Incidence, multiplicity, and size of liver tumors in trout fed diets containing E2, PFOA, PFNA, and PFDA were
significantly higher compared to AFB1-initiated animals fed control diet, whereas PFOS caused a minor increase in
liver tumor incidence. E2 and PFOA also enhanced MNNG-initiated hepatocarcinogenesis.

PFNA and PFDA enhanced incidence and size of liver tumors when compared with AFB1-initiated animals fed a
control diet. In the groups receiving PFNA or PFDA only, there were no tumors or a few liver adenomas, respectively.
This indicates a tumor promoting capacity of both compounds in this animal species.

Dose groups for initiation: 10 ppb AFB1
or 35 ppm MNNG, for both initiation
groups:
Cohort 1: diets containing 5 ppm E2,
2000 ppm PFOA(approximately 50 mg/kg
body weight/day), 2000 ppm FtOH, 1000
ppm PFNA, 200 ppm PFDA or 2000 ppm
CLOF ad libitum
Cohort 2: (AFB1 at 15 weeks), trout were
fed 100 ppm PFOS.
Finally, MNNG-initiated trout were fed 5
ppm E2 or 2000 ppm PFOA.
EFSA Opinion 2018: Risk to human health PFOS
• PFOS was found to cause tumours in the liver of rats. Mechanistic studies suggested that the compound may act as
related to the presence of PFOS and
a tumour promoter.
PFOA in food
PFOA
• In Sprague–Dawley rats, PFOA induced Leydig cell tumours. Hormonal dysbalance appears to be the underlying
mechanism. Inconsistent effects were noted for pancreatic hyperplasia or tumours in mammary gland and liver.

95

(EFSA,
2020b)