Document Bv8ZG26BDd0ook1J8nRmqGzRk

Proposal for reporting data relevant for human health Human Health Assessment of Perfluoroalkyl carboxylic Acids (PFCAs) 25/10/2022 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. 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 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 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 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. 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 [A3]: To put after experimental animal tox, organized by endpoint. Commented [A4]: Should we rather organize by endpoint and not substance? 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. 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 4 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). 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 [A11]: Should rather be based on EFSA and expanded with new studies. Put epidemiology after animal tox, cannot be divided by substance type 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. Commented [A13]: Add species Commented [A14]: This needs to be further described when toxicokinetic analysis is finished. 6 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 [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? 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 PPAR-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 PPAR-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 PPAR 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 PPAR 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 PPAR-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. 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? 8 5. 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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 a sensitive pubertal target in CD-1 and C57Bl/6 mice following perinatal perfluorooctanoic acid (PFOA) exposure. Reproductive Toxicology 54, 26-36. DOI: 10.1016/j.reprotox.2014.12.002 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 Vlez 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., Ernst E.H., Toft G. (2013): Associations of in utero exposure to perfluorinated alkyl acids with human semen 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- 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: 10.2131/jts.35.527 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 studies. PLoS One 6 (6), e20862. DOI: 10.1371/journal.pone.0020862 Zhang H., Hou J., Cui R., Guo X., Shi Z., Yang F., and Dai J. (2013): Phosphoproteome analysis reveals an important role for glycogen synthase kinase-3 in perfluorododecanoic acid-induced rat liver toxicity. Toxicol Lett 218 (1), 61-69. DOI: 10.1016/j.toxlet.2013.01.012 Zhang H., Shi Z., Liu Y., Wei Y., and Dai J. (2008): Lipid homeostasis and oxidative stress in the liver of male rats exposed to perfluorododecanoic acid. Toxicol Appl Pharmacol 227 (1), 16-25. DOI: 10.1016/j.taap.2007.09.026 Zheng F., Sheng N., Zhang H.X., Yan S.M., Zhang J.H., and Wang J.S. (2017): Perfluorooctanoic acid exposure disturbs glucose metabolism in mouse liver. Toxicology and Applied Pharmacology 335, 41-48. DOI: 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) "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)." 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 18 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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%) 92-day RDT study (OECD TG 408) Body weight ( -17% in HD males) Rat (Wistar) Liver: Abs. + rel. liver weight ( up to 33% in HD) n/sex/group = 10 (m+f) Hepatocellular hypertrophy (in up to 100% of HD rats) Haematopoietic system: Haemoglobin (females only, -8% in HD) Exposure: diet Mean corpuscular volume (females only, -6% in HD) 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) 98/1216 1043/- 9.9/12.2 98/123 9.9/123 98/1216 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 (Anonymo us, 2016; BayerCrop Science, 2014) 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) PFBA, 14-day RDT study, non-guideline study Liver: perfluorobuty to assess liver toxicity Catalase activity (marker enzyme of peroxisomes, +42%) - rate (purity Rat (SD) not specified), Induction of 80K-protein (associated with peroxisome proliferation) - C3F7CO2 n/sex/group = 3 (m) Exposure: oral (diet, 0.02%) Doses: 0, 20 mg/kg bw/d PFBA, perfluorobuty rate (98%), C3F7CO2 10-day RDT study, non-guideline study to assess liver toxicity Mouse (C57BL/6N) n/sex/group = 4 (m) Body weight ( +16%, but it was already significantly higher before treatment) Body weight gain ( +47% (+4.35% in Control, +6.38% in PFBA treatment, but no statistical analysis possible)) Liver Abs.+rel. liver weight ( +63% (abs.) or +38% (rel.)) Hepatic mitochondrial protein content ( +202%) 19 Body weight, 20 kidney, nervous 20 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 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%) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed chemistry Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) PFBA 18-day-ReproTox study (GD1-GD17) (ammonium Mouse (CD-1) salt, >98%) n/sex/group = 7-13 pregnant, 2-6 nonpregnant, 30-34 postweaning Exposure: oral (gavage) Doses: 0, 35, 175, 350 mg/kg bw/d 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) 175 175 20 Serum concentrations of PFBA (Das et al., in pregnant dams (g/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 (g/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 (g/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 (g/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 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) (Data also available for postweaning stages PD1 and PD10) PFBA, 28-day RDT study, non-guideline study Liver: 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- null, mice) not specified), humanized PPAR- transgenic mouse Hepatocellular focal necrosis with inflammatory cell infiltrate (no 35 C3F7CO2 model) statistical significance indicated but dose-dependent increase of incidences of mild cases) n/sex/group = 10 (m) Hepatocyte hypertrophy (no statistical significance indicated but - 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 Kidney, liver, At 35 mg/kg bw per day (24h (Foreman 35 nervous system, after final dosing) et al., immune system, Serum: 80 g/mL 175 haematopoietic system, Liver: 27 g/g 2009) endocrine 35 system, clinical 35 chemistry 35 Doses: 0, 35, 175, 350 mg/kg bw/d PFBA, 28-day RDT study, similar to OECD TG ammonium 407 perfluorobuty Rat (SD) rate (purity not specified), NH4C3F7CO2 n/sex/group = 10 (m+f) Exposure: oral (gavage) Doses: 0, 6, 30, 150 mg/kg bw/d Remarks: PFBA causes hepatomegaly and hepatocyte hypertrophy mediated via peroxisome proliferator-activated receptor- (PPAR) Liver: Abs. liver weight (m) ( +27% in MD, no effect after recovery) 6 Hepatocyte hypertrophy (no significance indicated but 60% incidence in HD and 0% in C and other dose groups) 30 Hepatic Acox, Cyp4A1, Cyp2B2, Malic (all m only) 6 Thyroid: Follicular hypertrophy/hyperplasia (no significance indicated but 90% incidence in MD and 30% in C) 6 Endocrine system: Serum total T4 (m) ( -59% in LD) - Serum free T4 (m) ( -46% in LD) - Clinical chemistry: Serum cholesterol (m) ( -20% in MD) 6 Immune system At 6 mg/kg bw/day in males (van 30 Serum: 24.7 17.6 g/mL Otterdijk, Liver: 7.5 4.5 g/g 2007a) 150 30 At 30 mg/kg bw/day in males Serum: 38.04 23.2 g/mL (Butenhoff Liver: 17.4 8.2 g/g et al., 30 2012a) 6 6 30 Remarks: During recovery, liver weight, histological, and cholesterol effects were resolved; mRNA transcript data for Acox and Cyp4A1 indicate activation of PPAR PFBA (purity 90-day RDT study, similar to OECD TG Liver: not specified), 408 Abs. liver weight (m) ( +23% in HD) 6 NH4C3F7CO2 Rat (SD) Hepatocyte hypertrophy (no significance indicated but 90% incidence in HD and 0% in C and other dose groups) 6 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 At 6 mg/kg bw per day in (van 30 males Otterdijk, Serum: 13.6 9.1 g/mL 2007b) 30 Liver: 3.1 2 g/g 30 At 30 mg/kg bw per day in (Butenhoff males 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, Substance study design, species, route of Observed effects (%), 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: Doses: 0, 1.2, 6, 30 mg/kg bw/d Serum total T4 (m) st, (. --39% in LD) NO(A)EL (mg/kg bw/d) 6 LO(A)EL (mg/kg bw/d) 30 6 30 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) Serum: 52.2 25 g/mL 2012a) Liver: 16.1 9.1gig Remarks: During recovery, liver weight, histological, and cholesterol effects were resolved; mRNA transcript data for Acox and Cyp4A1 indicate activation of PPARa PFBA 5-day RDT study No effect on gross or microscopic morphology of brain or spinal Rat cord Tbc 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 not specified) Mouse (ddY) n/sex/group = 3-5 (m+f) Body weight (m/f) (ns) Liver: Rel. liver weight '1' (m: ns; f, =+17% at LOEL) Peroxisomal 13-oxidation l' Exposure: i.p. Kidney, nervous system, immune 50 100 system, 50 haematopoietic system, thyroid, endocrine system (Kudo et al., 2006) Doses: 0, 50, 100, 150 mg/kg bw/d PFHxA (>99%) 28-day RDT study, similar to OECD TG 407 Rat (SD) n/sex/group = 10 (m+f) Exposure: oral (gavage; in 2% Tween 80/ deionized water) Doses: 0, 62.5, 125, 250, 500, 1000 mg/kg bw/d Body weight ,I, (m, ,- --16% at LOEL; f ns) Liver: .Abs. liver weight '1` (m/f, . +27%/+14% at LOEL) Rel. liver weight 1' (m/f, . +14%/+15% at LOEL) Hepatocellular hypertrophy 1' (m, 9 of 10 at 500 mg/kg bw/d) .Acyl-CoA oxidase 1' 1.-. +141% in males at 250 mg/kg bw/d) Kidney: 'Rel. kidney weight 1' (m/f, . +12%/+12% at LOEL) Immune system Abs. thymus weight .1, (m, =-27% at LOEL. Rel ns, f not measured) Spleen: increased extramedullidary hematopoiesis Thyroid weight Thyroid weight changes at all doses (m+f), but ns and not clearly dose related 500 250/250 125/250 250/500 250 250/500 500 250/500 -- 22 1000 500/500 250/M M no Plasma conc. (ng/ml) at 62.6 histopathological mg/kg bw/d at end of study: description for Thyroid gland 378 178 (ml 129 16 (f) Plasma conc. (ng/ml) at 250 mg/kg bw/d at end of study: (NTP, 2019b) Exp.no. C20613 == 1297 265 (m) 1000 500/1000 Liver conc (ng/g) at 250 mg/kg bw/d at end of study: 655 148 (m) 62.5 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) twice daily at one-half dose (total): 31.3 (62.6), 62.5 (125), 125 (250), 250 (500), 500 (1000) mg/kg bw/d Haematopoietic system: Erythrocytes (=-5%/-7% at LOEL), haemoglobin (=-3%/-6% at -/125 LOEL), haematocrit (,... --4%/-7% at LOEL) (m/f) 4, Endocrine system: .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` Serum cholesterol 4, (m, z.. -20% at LOEL) 250/250 Other effects: Degeneration and hyperplasia of olfactory epithelium (m+f) 125/125 LO(A)EL (mg/kg bw/d) 62.5/250 500/500 62.5 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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 Perfluorohexa developmental toxicity screening test, Mortality 1` (death of 5/15 m and 6/15 fin HD) 150 noic acid OECD TG 422 Body weight 4, 50 (98.5%), CAS: Rat (SD) Liver: 307-24-4/EC: Abs. + rel. liver weight (m/f) Is 50 206-196-6, C6HFu.O2 n/sex/group = 10-15 (m) Hepatocellular hypertrophy '1` (minimal (MD) to mild (HD) 50 Kidney: Exposure: oral (gavage), vehicle: deionized water Papillary necrosist 150 Immune system: 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 = 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, Urine samples collected from all animals 0-6, 6-12, 12-24 h following last dosing Half-life in serum and for urinary elimination about 2-3 h Proj. ID: WIL534001) (WIL, 2005) 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) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 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 NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed 150 300-450 150 300 150 300 150 300 150 300 - 50 PFHxA 90-day RDT study, similar to OECD TG Body weight gain (m) ( -7% in LD&HD, -10% in MD at day 90) 10 50 (98.5%) 408 Body weight gain (f) ( -5% in LD&HD at day 90, but ns) Rat (SD) Liver: Rel. liver weight (m) ( +22% in HD) (f ns) 50 200 n/sex/group = 10 (m+f) Centrilobular hepatocellular hypertrophy (m) Hepatic peroxisomal -oxidation (m) 50 200 50 200 Kidney: Exposure: oral (gavage) Rel. kidney weight (m) ( +8% in LD) Immune system - 10 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) 50 200 Reticulocyte ( +59% in HD) 50 200 Globulin (m) ( -8% in HD) 50 200 Clinical chemistry: Serum cholesterol (m) ( -26% in MD) (f ns 10 50 Triglycerides not measured Calcium (m) ( -3% in MD) 10 50 Serum ALT ( +237% in HD) and ALP (m) ( +109% in HD) 50 200 Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) (Chengelis et al., 2009) PFHxA 90-day RDT study, OECD TG 408, plus (sodium salt, 30 day recovery group 100%) Rat (Crl:CD(SD)) 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) Body weight (m) (-10% at day 90 at LOEL) Liver: Abs./rel. liver weight ( +63% in HD) Hepatocellular hypertrophy (no significance indicated but for males 40% incidence in MD, 100% in HD and 0% in C and LD) 100 100 20/100 24 500 500 100/500 (Loveless et al., 2009) 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) n/sex/group = 10 (m+f) Hepatic peroxisomal -oxidation (m/f) ( +85% in MD, +330% in20/100 HD) Kidney: Exposure: oral (gavage) Rel. kidney weight ( +16% in HD) 100 Urine volume ( +207% in HD) 100 Doses: 0, 20, 100, 500 mg/kg bw/d 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) LO(A)EL (mg/kg bw/d) 100/500 500 500 500 500 500 500 500 500 20 100 Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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)" PFHxA 110-day RDT study, as part of a one- Body weight (m) ( -12% at LOEL) 20 100 Liver, kidney, sodium salt (100%) generation reproduction study, OECD TG 415 Rat (Crl:CD(SD)) Body weight (f, GD0-7)) ( -31% at LOEL) 100 500 nervous system, immune system, haematopoietic system, thyroid, n/sex/group = 10 (m+f) endocrine system (Loveless et al., 2009) Exposure: oral (gavage) Doses: 0, 20, 100, 500 mg/kg bw/d 25 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFHxA Combined Developmental and Phase I ammonium salt (93.4%) perinatal/postnatal reproduction Body weight (ns) toxicity study Liver: ICH Harmonised Tripartite Guideline - Abs/rel liver weight (ns) S5(R2), stage C-F Mouse Crl:CD1(ICR), 69-76 d.o. at mating Phase II Body weight (slight reduction at mid and high dose on GD 18, lowest dose slightly higher than control) Liver: F0: 20 F/dose GDs 6-18(Males only Abs/rel liver weight (ns) used for breeding, no treatment) F1: 20/sex/dose (no direct treatment; observed until weaning to sexual maturity) Exposure: oral (gavage; in deionized water) Remark: Iwai and Hoberman 2014 abstract: "Based on these data, the maternal and reproductive no observable adverse effect level of PFHxA Ammonium Salt is 100 mg/kg bw/d." NO(A)EL (mg/kg bw/d) LO(A)EL (mg/kg bw/d) Key parameters / targets not addressed Postnatal functional parameters are not evaluated in this type of study Serum/tissue concentrateion of PFAS /metabolites (time of sampling) Reference (Iwai and Hoberman, 2014; Iwai et al., 2019) Study reports: Hoberman, 2011a (Phase I) Hoberman, 2011b (Phase II) Phase I: Doses: 0, 100, 350, 500 mg/kg bw/d Phase II Doses: 0, 7, 35, 175 mg/kg bw/d PFHxA 104-weeks Carcinogenic study Mortality (f only: 22% survival at 200 mg/kg bw/d vs 36% survival (98.1%) Combined chronic tox/carc study Rat Crl:CD(SD) in control, i.e. +100% mortality at 200 mg/kg bw/d, but no statistical testing) Body weight (no effect) Liver: n/sex/group = 60-70 Hepatocellular necrosis (weak increase at highest dose in m+f) Kidney: Necrosis (f only, minimal to severe papillary necrosis in 17 of 70 30 200 Male Dosage: 0, 2.5, 15, 100 mg/kg test animals and/or minimal to moderate renal tubular bw/d degeneration in 7 of 70 test animals) Female Dosage: 0, 5, 30, 200 mg/kg Urinary parameters: mean urine volume (f); slightly lower specific30 200 bw/d 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) 30 200 Heamoglobin (-5% after 51 weeks only in females) 30 200 Clinical chemistry: 26 (Klaunig et al., 2015) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Serum triglycerides (m: -43% at2.5 and 100, but not at 15 mg/kg bw/d after 52 weeks, authors conclude no effect) Serum LDL (f: -44% at LOEL after 26 weeks, but not after 52 weeks) Serum HDL (ns) NO(A)EL (mg/kg bw/d) - 30 LO(A)EL (mg/kg bw/d) 2.5 200 Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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) PFHpA, 5-day RDT study, non-guideline study, Perfluorohept to assess hepatic peroxisomal - anoic acid oxidation through PFCAs (purity not Rat (Wistar) specified), CAS: 375-85-9 n/sex/group = 4 (m+f) / EC: 206-798- 9, C7HF13O2 Exposure: i.p. Liver: hepatic peroxisomal -oxidation (m/f) 30/160 Remarks: potency of the induction of peroxisomal -oxidation was compared between PFCAs; highly significant correlation between the induction and hepatic concentrations of PFCAs in the liver regardless of their carbon chain lengths 160/- kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry concentrations of PFHA in the (Kudo et liver were below detection al., 2000) limit <3 g:g liver Doses: 0, 30, 160 mg/kg bw/d PFHpA (purity 5-day RDT study, non-guideline study, not specified) Mouse (ddY) n/sex/group = 3-5 Liver: Abs. and rel. liver weight (m/f, +68% in MD) Peroxisomal -oxidation Activity of hepatic microsomal 1-Acyl-GPC acyltransferase (m, only HD tested) 20/50 -/20 - Exposure: i.p. Doses: 0, 20, 50, 100 mg/kg bw/d PFHpA (purity 3-day RDT study, non-guideline study not specified) Mouse (C57BL/6) Remarks: the longer the perfluoroalkyl chain, the more PFCA accumulates in the liver; accumulated PFCAs induce hepatomegaly, peroxisomal -oxidation and microsomal 1-acyl-GPC acyltransferase Liver: Hepatic mRNA levels of Cyp4a10 - n/sex/group: 4 Remarks: Mechanistic study to assess the MoA of PFCAs Exposure: i.p. 27 50/100 20/50 100 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry kidney, nervous 20 system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry (Kudo et al., 2006) (Abe et al., 2017) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) Doses: 0 , 20 mg/kg bw/d PFHpA as combined 90-day RDT study with Liver: Sodium reproduction/developmental toxicity Centrilobular hypertrophy of hepatocytes perfluorohept screening (similar to OECD TG 408 and Abs. and rel. liver weight anoate 422) Clinical chemistry: (>99.3%) Mouse (CD1) exposure time F0, m: 90d prior to mating (total 109-113 d); F0, f: 90d prior to pairing and until lactation ALT levels in lactating females (D21) ALP, ALT and Trig. in m and in ALP and Trig. in non-mated f Endocrine system: Thyroid T4 levels in serum (m only) (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 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 Doses: 0, 0.5, 10, 50 mg/kg bw/d Adrenal rel. and abs. weights in f in HD -/- 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 (Anonymo us, 2017) 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 Liver: kidney, nervous Perfluoroocta Rat (Wistar) noic acid Peroxisomal -oxidation (m/f) 5/20 10/- system, immune system, (analytical grade), EC: n/sex/group = 4 Remarks: Mechanistic study to assess the MoA and to investigate haematopoietic system, thyroid, 206-397-9, influence of chain length; peroxisomal -oxidation is statistically endocrine CAS: 335-67- Exposure: i.p. injection (vehicle: 1, C8HF15O2 propyleneglycol:water (1:1, v:v) highly correlated with PFCA concentration in the liver (r=0.850, p<0.001) internal dose in liver decisive for effect, not carbon chain length or sex system, clinical chemistry (Kudo et al., 2000) Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d PFCAs assessed: PFHpA, PFOA, PFNA, 28 Commented [A22]: How important is it to fill in this? Most relevant for guideline studies deviating form guideline? 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFDA NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) PFOA (purity 5-day RDT study, non-guideline study Liver: not specified) Mouse (ddY) Abs. and rel. liver weight (m/f) ( +39% in LD) -/- Peroxisomal -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 Remarks: "(...) the longer the perfluoroalkyl chain, the more PFCA accumulates in the liver (... and) accumulated PFCAs induce hepatomegaly, peroxisomal -oxidation and microsomal 1-acyl-GPC acyltransferase (...)"(from abstract; refers to C6-C9) 2.5/2.5 2.5/2.5 20 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry 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) PFOA (purity 3-day RDT study, non-guideline study Liver: not specified) Mouse (C57BL/6) Rel. liver weight ( +100%) - Hepatic mRNA levels of Cyp4a10, Acox1, Cyp2b10, Aldh1 - n/sex/group = 4 Remarks: Mechanistic study to assess the MoA of PFCAs Exposure: i.p. kidney, nervous 20 system, immune 20 system, haematopoietic system, thyroid, endocrine system, clinical chemistry (Abe et al., 2017) Doses: 0 , 20 mg/kg bw/d PFOA (>96%) 7-days RDT study, non-guideline study Body weight - 1 kidney, nervous Mouse (Balb/c) Liver: Abs. liver weight ( +60% at LOAEL) 1 5 system, immune system, n/sex/group = not reported Necrosis and vacuolation of hepatocytes Hepatic triglycerides - 1 - 1 haematopoietic system, thyroid, mRNA levels of fatty acid translocase and lipoprotein lipase - 1 endocrine system Exposure: oral (vi distilled water) Clinical chemistry: ALT ( +80% at LOAEL) - 1 Serum fatty acids ( -62% at LOAEL) 1 5 Doses: 0, 1, 5 mg/kg bw/d Serum triglycerides ( -58% at LOAEL) - 1 (Hui et al., 2017) Remarks: Mechanistic study to assess the MoA of PFCAs Commented [A22]: How important is it to fill in this? 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Formatted: French (France) 29 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFOA (purity 14-days RDT study, non-guideline Body weight not specified) study Liver: Rat (SD) Abs. + rel. liver weight Activity of hepatic superoxide dismutase and glutathione n/sex/group = 7 (m only) peroxidase MDA content in liver Expression of PPAR mRNA Exposure: oral (gavage) Expression of CYP4A1 mRNA PPAR protein NO(A)EL (mg/kg bw/d) 5 LO(A)EL (mg/kg bw/d) 25 1 5 - 1 1 5 - 1 - 1 - 1 Key parameters / targets not addressed kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system Serum/tissue concentrateion of PFAS /metabolites (time of sampling) Reference (Wang et al., 2017) Article in Chinese, only abstract in English PFOA (96%) Doses: 0, 1, 5, 25 mg/kg bw/d 21-days RDT study, non-guideline study Mouse ( Balb/c) n/sex/group = 3-10 (m only) Remark: CYP4A1 is a PPAR-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%) - Exposure: oral (gavage) Doses: 0, 1.25 mg/kg bw/d 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 Liver: (ammonium study Abs. and rel. liver weight 1 salt, <98%) Mouse (Kunming) Hepatic functional enzymes (GPT, GOT) 1 Hepatic triglycerides ( +150% in HD) 1 Visible vacuoles around liver portal area 1 n/sex/group: 8 (m only) Hepatic FGF21 protein 1 Clinical chemistry: Exposure: oral (gavage, in peanut oil + Serum cholesterol ( -26% in MD) 1 0.5% DMSO) Serum glucose ( -25% in HD) 1 Serum insulin ( -45% in HD) 1 Serum triglyceride ( -57% in HD) 1 Doses: 0, 1, 5 mg/kg bw/d Serum L-LDL ( +140% in HD) 1 Serum H-LDL ( -50% in HD) 1 Other effects: Insulin-positive cells in pancreatic islets 1 PFOA 2, 8 or 16 weeks RDT study, non- Body weight ( -22 - -37%) - (ammonium guideline study Liver: salt, >98%) Mouse (57BlL/6) Abs./rel. liver weight ( +50%) - 30 kidney, nervous PFOA level in liver: 125.9 (Zheng et 1.25 system, immune 10.0 g/g al., 2017) 1.25 system, 1.25 hsyasetmemat,otphoyrieotiidc, PFOA level in serum: 55.5 1.25 endocrine system 0.50 g/mL 1.25 kidney, nervous 5 system, immune 5 system, 5 haematopoietic 5 system, thyroid, 5 endocrine system 5 5 5 5 5 5 5 1 kidney, nervous system, immune 1 system, (Wu et al., 2018) (Li et al., 2019) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks n/sex/group = 5 (m only) Exposure: oral (gavage, in distilled water) Replication of hepatocytes (week 2+8) (4.3-fold increase of the hepatocytes DNA synthesis Hepatic peroxisomal -oxidation activity (week 2-16) Activation of PPAR Expression of fatty acid metabolism genes Remarks: Mechanistic study to assess the MoA NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) - 1 - 1 - 1 - 1 Key parameters / targets not addressed haematopoietic system, thyroid, endocrine system, clinical chemistry Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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) 340 37/115 -/37 115/- Remarks: Acute oral toxicity (LD50 540 mg/kg), primary skin irritation Doses: 0, 30, 100, 300, 1000, 3000, (no effects), eye irrigation (moderate) and one-hour inhalation data 10000, 30000 ppm (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) 1600 115/340 -/115 340/- kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry (Griffith and Long, 1980; Study report by Metrick and Marisa, 1977) PFOA ("FC143" (3M), ammonium salt, purity not specified) 28-day RDT study Mouse (albino) 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) 760 240 13/51 13 13 13/- - 13/- - - - 1900 760 51/240 51 51 51/13 13 240/13 13 13 13 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry (Study report by Christophe r and Marias, 1977; Griffith and Long, 1980) 31 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks slightly higher than males) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) PFOA (ammonium salt) 90-day RDT study Rat (ChR-CD albino), 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 7.2/- 7.2/22 7.2/- 75 22- 22/75 7.2/- nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical chemistry Fluorine in pooled (n=4-5) serum (ppm) (m/f) 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) Doses: 0, 10, 30, 100, 300, 1000 ppm (equivalent to 0.7, 2.2, 7.2, 22, 75 mg/kg bw/d, female doses slightly higher than males) ND = None detected NM = Not measured PFOA (ammonium salt) 90-day RDT study Rhesus monkey, n/sex/group = 2 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 Exposure: oral (gavage) Doses: 0, 3, 10, 30, 100 mg/kg bw/d 10 30 body weight, Ammonium PFOA in serum (Study liver, kidney, (ppm) (m/f) report by nervous system, immune system, haematopoietic system, thyroid, endocrine system, clinical 0 mg/kg bw/d: NM/1 3 mg/kg bw/d: 48.53/50.65 10 mg/kg bw/d: 45.71/71.79 30 mg/kg bw/d: 145/NM 100 mg/kg bw/d: NM/NM Goldenthal , 1978b; Griffith and Long, 1980) chemistry 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 Body weight (ns) kidney, nervous (ammonium Rat (Crl:CDBR) salt) Liver: Abs. liver weight ( +36%% at LOEL in week 8) 0.06 0.65 system, immune system, n/sex/group = 10-25 (males only) Rel. liver weight ( +37% at LOEL in week 8) Hepatocellular hypertrophy 0.06 0.65 0.06 0.65 haematopoietic system, thyroid, Hepatic palmitoyl CoA oxidase activity ( +133% at LOEL after in 0.65 1.94 endocrine Exposure: oral (dietary) week 14) system, clinical chemistry (Palazzolo, 1993) 32 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) Doses: 0, 0 (pair-fed to 100 ppm), 1, Remark: Effects seemingly reversible. Overall, NOAEL is 100 ppm (6.5 10, 30, 100 ppm mg/kg bw/d), NOEL is 1 ppm (0.06 mg/kg bw/d) LO(A)EL (mg/kg bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) equivalent to 0, 0.06, 0.65, 1.94, 6.5 mg/kg bw/day (calculated in study report) PFOA (ammonium salt, 95.2% purity) 26 weeks + recovery (2 control and 2 Body weight mid-dose monkeys) 13 weeks more 30 mg/kg bw/d (in first 11 days) -3 to -7.5% (no food consumption), 10 Cynomolgus monkey 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 n/sex/group = (males only) steep increase in body weight but overall weight still reduced. Recovery animals from 10 mg/kg bw/d group much lower body - weight gain in 14 weeks after treatment Exposure: oral (gelatine capsules) Liver: 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) 20-30 10 3 20-30 3 10 20-30 10 kidney, nervous system, immune system, haematopoietic system, thyroid Liver tissue PFOA concentration (g/g) (values of individuals, mean sd in bold)) Control: 0.09, 0.23, <LOQ (2x) 3 mg/kg bw/d: 15.2, 18.5, 11.3, 18.3 (15.8 3.4) (Butenhoff et al., 2002; Study report by Thomford, 2001) 10 mg/kg bw/d: 21.9, 6.3, 8.9, 18.8 (14.0 7.6) 30/20 mg/kg bw/d: 16, 83.3 Control recovery: <LOQ (2x) 10 mg/kg bw/d recovery: 0.15, 0.08 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 3 Nervous system, (ammonium cohabilitation), 2-generation guideline Liver: immune system, salt, 97.99%) study EPA OPPTS 8703800 Abs.+rel. liver weight ( +20% at LOEL) - 1 haematopoietic Kidney: system, thyroid, Rat (Sprague-Dawley) Rel. kidney weight ( +16.5% at LOEL) - 1 endocrine system, clinical n/sex/group = 30 (males only) chemistry (Butenhoff et al., 2004) 33 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Exposure: oral (gavage) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) Doses: 0, 1, 3, 10 mg/kg bw/d PFOA 7 day-study Liver: (ammonium Mouse (PPAR-null mice (129S4/SvJae- Abs. liver weight ( +30% at LOEL) 0 salt, purity) Pparatm1Gonz/J) and wildtype Rel. liver weight ( +32% at LOEL) 0 (WT)(129S1/SvlmJ - SV/129 and CD-1)) Hepatocyte hypertrophy (lesion score) ( +73% at LOEL) 0 Labelling index (LI) ( +3750% at LOEL) 3 n/sex/group = (m only) Exposure: oral (gavage), PFOA in deionized water Doses: 0, 1, 3 mg/kg bw/d Peroxisome proliferation Increased cell proliferation (only in wild-type) Primary effects of PFOA on WT mice was on genes associated with fatty acid metabolism, consistent with activation of PPAR Other alterations on genes related to inflammation (complement/coagulation cascades), and xenobiotic metabolism Altered expression of genes associated with cell cycle control, peroxisome biogenesis and proteasome structure/organization Cyp2b and Cyp2c genes upregulated Remark: "In wild-type mice, PFOA (...) induced changes consistent with activation of PPAR. (...)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 PPAR." (from abstract of Rosen et al. 2008) Body weight, Serum concentrations of PFOA (Wolf et 1 kidney, nervous in WT/CD-1/KO mice (g/ml): al., 2008) 1 system, immune Control: 0.012 0.006/ 0.032 (histopath 1 system, 0.023/ 0.010 0.004 ology) 10 haematopoietic 1 mg/kg: 14.1 4.3/17.2 7.3/(Rosen et system, thyroid, 17.7 4.4 al., 2008) endocrine system, clinical 3 mg/kg: 33.3 15.0/ - / 47.9 (Gene 18.9 expression) chemistry 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 (g/ml): 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 PFOA 14-day-study Body weight (day 7) ( -17% at LOEL) 10 30 nervous system, Serum PFOA concentrations (Loveless (ammonium Rat (Crl:CD(SD)IGS BR) Body weight (day 13) ( -19% at LOEL) 10 30 salt, Body weight gain (0-13) ( -45% at LOEL) 3 10 linear/branch Liver: ed (80:20) n/sex/group = 10 (m only) Abs. liver weight ( +60% at LOEL) 1 3 isoforms) Rel. liver weight ( +15% at LOEL) 0.3 1 Exposure: oral (gavage, in water) Hepatic peroxisomal -oxidation activity ( +48% at LOEL) 0.3 1 Kidney: Abs. kidney weight ( +11% at 3 mg/kg bw/d, -12% at 30 mg/kg - - immune system, (g/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 et al., 2006) Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg bw/d) bw/d Rel. kidney weight (+9% at LOEL) 1 3 Haematopoietic system: Haemolysis in vena cava 1 3 Haemolysis in orbital sinus 0 0.3 34 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Clinical chemistry: Serum cholesterol ( -55% at LOEL) Serum HDL ( -48% at LOEL) Serum non-HDL ( -43% at LOEL) Serum triglycerides ( -34% at LOEL) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 1 3 1 3 0.3 1 0.3 1 PFOA 14-day-study Body weight (day 7) ( -31% at LOEL) 10 30 nervous system, Serum PFOA concentrations (Loveless (ammonium salt, linear isoforms) Rat (Crl:CD(SD)IGS BR) n/sex/group = 10 (m only) Body weight (day 13) ( -19% at LOEL) Body weight gain (0-13) ( -26% at LOEL) Liver: Abs. liver weight ( +28% at LOEL) 10 30 1 3 1 3 Rel. liver weight ( +14% at LOEL) 0.3 1 Exposure: oral (gavage, in water) Hepatic peroxisomal -oxidation activity ( +90% at LOEL) 0.3 1 Kidney: Abs. kidney weight ( -16% at LOEL) 10 30 immune system, (g/ml): haematopoietic 0 mg/kg: 0.39 0.51 g/ml system, thyroid, 0.3 mg/kg: 20 3.2 endocrine system 1 mg/kg: 65 11 3 mg/kg: 137 18 10 mg/kg: 206 65 30 mg/kg: 223 77 et al., 2006) Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg Haematopoietic system: bw/d Haemolysis in vena cava 1 3 Haemolysis in orbital sinus 0.3 1 Clinical chemistry: Serum cholesterol ( -27% at LOEL) 0 0.3 Serum HDL ( -42% at LOEL) 1 3 Serum non-HDL ( -32% at LOEL) 0 0.3 Serum triglycerides ( -21% at LOEL) 0 0.3 Remark: Conclusion of the study: Linear PFOA is similarly toxic as linear/branched-mix (80:20), branched isoforms have weaker toxicity PFOA 14-day-study Body weight gain (0-13) ( -24% at LOEL) 10 30 (ammonium Rat (Crl:CD(SD)IGS BR) salt, branched isoforms) n/sex/group=10 (m only) Liver: Abs. liver weight ( +19% at LOEL) 0.3 1 Rel. liver weight ( +16% at LOEL) 0.3 1 Hepatic peroxisomal -oxidation activity ( +34% at LOEL) 1 3 Kidney: Exposure: oral (gavage, in water) Rel. kidney weight ( +10% at LOEL) Haematopoietic system: 3 10 Haemolysis in vena cava 3 10 Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg Haemolysis in orbital sinus 1 3 bw/d Clinical chemistry: Serum cholesterol ( -22% at LOEL) 0.3 1 Serum HDL ( -30% at LOEL) 1 3 Serum non-HDL ( -50% at LOEL) 1 3 Serum triglycerides ( -44% at LOEL) 3 10 nervous system, Serum PFOA concentrations immune system, (g/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) 35 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFOA 14-day-study Body weight (day 7) ( -16% at LOEL) (ammonium Mouse (Crl:DC-1(ICR)BR) salt, linear/branch ed (80:20) n/sex/group = 10 (m only) isoforms) Exposure: oral (gavage, in water) 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 -oxidation activity ( +92% at LOEL) Kidney: Abs. kidney weight ( -20% at LOEL) Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg Clinical chemistry: bw/d Serum cholesterol ( -33% at LOEL) Serum HDL ( -43% at LOEL) Serum triglycerides ( +37% at LOEL) NO(A)EL (mg/kg bw/d) 10 3 3 LO(A)EL (mg/kg bw/d) 30 10 10 1 3 1 3 0 0.3 10 30 Key parameters / targets not addressed nervous system, immune system, haematopoietic system, thyroid, endocrine system Serum/tissue concentrateion of PFAS /metabolites (time of sampling) Serum PFOA concentrations (g/ml): 0 mg/kg: 0.04 0.02 0.3 mg/kg: 10 1.4 1 mg/kg: 27 5 3 mg/kg: 66 8.6 10 mg/kg: 190 29 30 mg/kg: 241 28 Reference (Loveless et al., 2006) 1 3 1 3 0 0.3 PFOA 14-day-study Body weight (day 7) ( -16%) 10 30 nervous system, Serum PFOA concentrations (Loveless (ammonium salt, linear isoforms) Mouse (Crl:DC-1(ICR)BR) n/sex/group = 10 (m only) Body weight (day 13) ( -18%) Body weight gain (0-13) ( -500%) Liver: Abs. liver weight ( +61% at LOEL) 10 30 10 30 0.3 1 Rel. liver weight ( +17% at LOEL) 0 0.3 Exposure: oral (gavage, in water) Hepatic peroxisomal -oxidation activity ( +143% at LOEL) 0 0.3 Kidney: Abs. kidney weight ( -18% at LOEL) 3 10 immune system, (g/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 3 mg/kg: 69 10 10 mg/kg: 225 68 30 mg/kg: 259 34 et al., 2006) Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg Rel. kidney weight ( -11% at LOEL) 3 10 bw/d Clinical chemistry: Serum cholesterol ( -35% at LOEL) 1 3 Serum HDL ( -46% at LOEL) 1 3 PFOA 14-day-study Body weight (day 13) ( +6%) 1 3 nervous system, Serum PFOA concentrations (Loveless (ammonium Mouse (Crl:DC-1(ICR)BR) salt, branched isoforms) n/sex/group = 10 (m only) Body weight gain (0-13) ( +200%) Liver: Abs. liver weight ( +149% at LOEL) Rel. liver weight ( +19% at LOEL) 1 3 1 3 0 0.3 Hepatic peroxisomal -oxidation activity ( +103% at LOEL) 0.3 1 Exposure: oral (gavage, in water) Clinical chemistry: Serum cholesterol ( -34% at LOEL) 1 3 Serum HDL ( -34% at LOEL) 1 3 immune system, (g/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 3 mg/kg: 82 10 10 mg/kg: 172 29 30 mg/kg: 244 50 et al., 2006) Doses: 0, 0.3, 1, 3, 10, or 30 mg/kg Serum non-HDL ( -35% only at 3 mg/kg) - - bw/d 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) (ammonium Immunotoxicity, Health Effects Test Body weight gain over 28 days ( -26% at LOEL) 1 10 Kidney, nervous 1 10 system, (Loveless et al., 36 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks salt, linear) Guidelines (1998) Liver: 19.5% aqueous Rat (Crl:CD(SD)IGS BR) Abs. liver weight ( +30%% at LOEL) Rel. liver weight ( +83% at LOEL) solution (in NANOpure n/sex/group = 10 (m only) Immune system: Rel. thymus weight ( +44% at LOEL) water) Abs./rel. spleen weight (ns) Exposure: oral (gavage) Anti-sRBC IgM antibody (ns) 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 solution (in Mouse (Crl:CD-1(ICR)BR) Abs. spleen weight ( -44% at LOEL) Rel. spleen weight ( -14% at LOEL) NANOpure Abs. + rel. thymus weight ( -45% at LOEL) water) n/sex/group=20 (m only) Anti-sRBC IgM antibody ( -22% at LOEL) Endocrine system: Exposure: oral (gavage) Serum corticosterone ( +120% at 10 mg/k/d) Clinical chemistry: Cholesterol ( -31% at LOEL) Doses: 0, 0.3, 1, 10, 30, 30/0 mg/kg bw/d HDL ( -29% at LOEL) Non-HDL (ns) Triglycerides ( -53% at LOEL) PFOA (90%) 3-week study, non-guideline Liver: Mouse (no strain given) Abs. liver weight ( +77% at LOEL) Total glutathione ( +101% at LOEL) n/sex/group = 5 (f only) Hepatic catalase ( +482% at LOEL) Hepatic SOD (ns) exposure: oral (gavage, PFOA in water) Hepatic Glutathione reductase ( +156% at LOEL) doses: 0, 1, 5, 10 mg/kg bw/d NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) 0.3 1 1 10 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) haematopoietic 2008) system, thyroid 10 30 (30/0) - 0.3 - 0.3 1 10 Kidney, nervous system, 0.3 1 haematopoietic system, thyroid 1 10 0.3 1 1 10 1 10 1 10 0.3 1 1 10 1 5 1 5 1 5 10 5 10 Body weight, kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Loveless et al., 2008) (Ahmed and Abd Ellah, 2012) PFOA (99.9%) 14-days, rat (CD) 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 10 kidney, nervous 1 10 system, immune system, 1 10 haematopoietic (Cook et al., 1992) 37 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Rel. liver weight ( +181% at LOEL) Exposure: oral (gavage) Hepatic -oxidation activity ( +246% at LOEL) Endocrine system: Doses: 0, 1, 10, 25, 50 mg/kg bw/d Serum estradiol ( +64% at LOEL) Reproductive tissue: Abs./rel. testes weight ( +20%/+21% at LOEL) Abs. accessory sex organ weight ( -16% at LOEL) Abs. ventral prostate weight ( -12% at LOEL) NO(A)EL (mg/kg bw/d) 1 1 LO(A)EL (mg/kg bw/d) 10 10 Key parameters / targets not addressed system, thyroid, 1 10 10 25 10 25 25 50 PFOA (99.9%) 14-days Rat (Crl:CD BR(CD)) n/sex/group = 15 (m only) Exposure: oral (gavage) Doses: 0, 25 mg/kg bw/d Body weight ( -16%) Liver: Rel. liver weight ( +79%) Hepatic -oxidation ( +760%) Hepatic aromatase ( +354%) Endocrine system: Serum estradiol ( +184%) Reproductive tissue: Testicular interstitial fluid testosterone ( +93%) Testicular interstitial fluid estradiol ( +115%) kidney, nervous system, immune system, haematopoietic system, thyroid, PFOA (98- 24-months-study Body weight gain (ns) 100%) Rat (Crl:CD BR (CD)) Liver: Liver adenoma ( +1200% (13/1)) n/sex/group = 156 (m only) Endocrine system: Estradiol ( +271/+110/+70/+71/+78% after 1/3/6/9/12 month(s) n/group (hormonal analysis) = 10 Prolactin ( -40/-29% after 3/6 months) n/group (peroxisome prolif.) = 6 Reproductive tissue: Testis weight ( +8% after 24 months) Leydig cell adenoma ( +267%) Exposure: oral (dietary) Other effects: Pancreatic cell proliferation ( +175/+125% after 15/18 months) Doses: 0, 300 ppm (equivalent to 13.6 Pancreatic acinar cell hyperplasia ( +290%) mg/kg bw/d) Pancreatic acinar cell adenoma ( +800%) Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) (Biegel et al., 1995) (Biegel et al., 2001) 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 20 , nervous system, Blood PFOA concentrations (Cui et al., immune system, (g/ml): 2009) - 5 - 5 - 5 - 5 haematopoietic system, thyroid 0 mg/kg: 0 5 mg/kg: 39.2 14.4 20 mg/kg: 58.8 17.6 Liver PFOA concentrations 38 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 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 NO(A)EL (mg/kg bw/d) - LO(A)EL (mg/kg bw/d) 5 Key parameters / targets not addressed - 5 - 5 - 5 Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) (g/g): 0 mg/kg: 0 5 mg/kg: 218 21 20 mg/kg: 196 10 Kidney PFOA concentrations (g/g): 0 mg/kg: 0 5 mg/kg: 228 37 20 mg/kg: 209 74 Lung PFOA concentrations (g/g): 0 mg/kg: 0 5 mg/kg: 63.0 11.3 20 mg/kg: 64.3 15.9 Heart PFOA concentrations (g/g): 0 mg/kg: 0 5 mg/kg: 35.5 17.6 20 mg/kg: 34.6 18.0 Spleen PFOA concentrations (g/g): 0 mg/kg: 0 5 mg/kg: 13.6 2.4 20 mg/kg: 6.92 9.31 Testicle PFOA concentrations (g/g): 0 mg/kg: 0 5 mg/kg: 16.7 16.9 20 mg/kg: 16.8 19.2 Brain PFOA concentrations (g/g): 0 mg/kg: 0 39 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed PFOA (97%) 7 days Rat (SV129 WT and PPAR-null) n/sex/group = 4 (m only) Exposure: oral (gavage) Body weight (ns) Liver: Abs. liver weight (WT/PPARA-null) ( +170%/+175%) Rel. liver weight (WT/PPARA-null) ( +143%/+171%) Cell area (WT/PPARA-null) ( +105%/+96%) Hepatic DNA content (WT/PPARA-null) ( -43%/-35%) Hepatic lipid accumulation (WT) ( +900%) Hepatic triglycerides (WT) ( +225%) kidney, nervous system, immune system, haematopoietic system, thyroid Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 5 mg/kg: 10.5 9.8 20 mg/kg: 7.20 6.03 (Das et al., 2017) Doses: 0, 10 mg/kg bw/d PFOA (98%) 15 days, non-guideline study Mouse (C57BL/6N) n/sex/group = 8 (f only) Exposure: oral (drinking water) 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) Doses: 0, 0.94, 1.88, 3.75, 7.5, 15, 30 mg/kg bw/d 7.5 15 Liver, kidney, Serum PFOA concentrations (DeWitt et nervous system, (ng/mL) at 1 day postdosing: al., 2008) 1.88 3.75 7.5 15 1.88 3.75 1.88 3.75 haematopoietic system, thyroid 0 mg/kg: 54.3 4.9 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: 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 PFOA (98%) 10-day-study Body weight (sham) ( -9.8% at LOEL at 2 days postdosing) 3.75 7.5 Liver, kidney, Mouse (C57BL/6N; adx/shamadrenolectomized) Body weight (adx) ( -10.3% at LOEL, at 2 days postdosing) 7.5 15 Immune system: SRBC IgM titer (sham) ( -18% at LOEL) 7.5 15 nervous system, haematopoietic system, thyroid n/sex/group = 5-7 (f only) SRBC IgM titer (adx) ( -14% at LOEL) Endocrine system: 3.75 7.5 Exposure: oral (drinking water) Serum corticosterone ( +168% at LOEL) 7.5 15 Clinical Chemistry (after 5 days of dosing, no effects after 10 days) Doses: 0, 3.75, 7.5, 15 mg/kg bw/d ALT (adx) ( +250% at LOEL) AST (adx) ( +72% at LOEL) 7.5 15 7.5 15 SDH (adx) ( +135% at LOEL) 7.5 15 Cholesterol (sham) ( -19% at LOEL) 7.5 15 Triglycerides (sham) ( -45% at LOEL) - 3.75 (DeWitt et al., 2009) 40 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Triglycerides (adx) ( -60% at LOEL) LDH (adx) ( +36% at LOEL) PFOA 15-day-study (immunization on day 11) Body weight ( -14% at LOEL from day 12 onwards, only in WT) (ammonium salt, 98%) Mouse (WT (C57BL/6-Tac)) and PPAR Immune system: KO (B6.129S4-Ppartm1GonzN12)) Rel. spleen weight (only WT) ( -32% at LOEL) Rel. thymus weight (only WT) ( -58% at LD, no effect at HD) n/sex/group = 6 (f only) T-cell dependent IgM antibody response (WT) ( -17%) T-cell dependent IgM antibody response (PPAR-KO) ( -14%) NO(A)EL (mg/kg bw/d) 3.75 7.5 7.5 7.5 7.5 7.5 LO(A)EL (mg/kg bw/d) 7.5 15 30 30 30 30 Exposure: oral (drinking water) Doses: 0, 7.5, 30 mg/kg bw/d Remark: Suppression of T-cell dependent antibody response seemed to be PPAR-independent. "...effects on humoral immunity are likely mediated by disruption of B-cell/plasma cell function." Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) Liver, kidney, nervous system, haematopoietic system, thyroid, endocrine system, clinical chemistry (DeWitt et al., 2016) PFOA 15-day-study (immunization on day Body weight (ns) Liver, kidney, (ammonium 11), mouse (WT (C57BL/6N)) Immune system: nervous system, salt, 98%) n/sex/group = 8 (f only) Rel. spleen weight ( -17% at LOEL) 3.75 7.5 Rel. thymus weight ( -14% at LOEL) 3.75 7.5 Exposure: oral (drinking water) T-cell-independent IgM antibody response ( -10% at LOEL) 0.94 1.88 Doses: 0, 0.94, 1.88, 3.75, 7.5 mg/kg haematopoietic system, thyroid, endocrine system, clinical bw/d chemistry (DeWitt et al., 2016) PFOA (ammonium salt, 98%) 15-day-study (immunization on days 11) Mouse (WT (C57BL/6N)) n/sex/group = 4 (f only) Exposure: oral (drinking water) Doses: 0, 3.75, 7.5 mg/kg bw/d 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) Remark: there were no PFOA effects before immunization on day 10 body weight, liver, kidney, nervous system, haematopoietic 3.75 system, thyroid, clinical chemistry (DeWitt et al., 2016) 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), - kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Haughom and Spydevold, 1992) 41 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 0.02% (equivalent to 17 54% for Glucose-6-phosphate dehydrogenase) mg/kg bw/d) Acetyl-CoA Cholesterol acyltransferase (ACAT) activity) ( -51%) Clinical chemistry: Serum cholesterol ( -56%) Serum triglycerols ( -39%) NO(A)EL (mg/kg bw/d) LO(A)EL (mg/kg bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) Remark: Also PFOSA data available PFOA 28-day-studies (study 1 and 2) Body weight ( -12%/-11% on day 8; -23%/-13% on day 29) kidney, nervous Plasma levels of APFO (Elcombe (ammonium Rat (Sprague-Dawley) salt, 98%) n/sex/group = 10 (m only) 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) system, immune (g/mL): system, Control: < quantification limit haematopoietic system, thyroid, Day2: 259 39 endocrine system Day 8: 234 33 et al., 2010) Exposure: oral (dietary) Hepatic cell proliferation ( +236%/+114% on day 2; +318%/+413% on day 8; +164% on day 29 (only study 2 data Day 29: 252 45 available) Doses: 0, 300 ppm (equivalent to 20 Relative liver DNA concentration ( -13% on day 2 (only study 1 mg/kg bw/d) 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) PFOA 7-day RDT study Liver: Body weight, (Eldasher (ammonium Mouse (C57BL/6) salt) Abs. + rel. liver weight ( +35% at LOEL) Hepatic mRNA/protein expression of Cyp4a14 - 1 - 1 nervous system, immune system, et al., 2013) n/sex/group = 6 (m only) ( +11900%/+1150% at LOEL) Hepatic mRNA/protein expression of Bcrp ( +90%/+70% at LOEL)- 1 haematopoietic system, thyroid, Kidney: endocrine system Exposure: oral (gavage) Renal mRNA/protein expression of Cyp4a14 ( +90%/ns at LOEL) 1 3 PFOA (ammonium salt, >96%) Doses: 0, 1, 3 mg/kg bw/d 10-day RDT study (hypersensitivity study) Mouse (BALB/c) Liver: Abs. liver weight ( +53% at LOEL) Immune system: Thymus weight ( -41% at LOEL) 42 Body weight, 2.5 6.25 kidney, nervous system, 25 50 haematopoietic (Fairley et al., 2007) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks n/sex/group= 5 (f only) Spleen weight ( -28% at LOEL) Exposure: dermal (PFOA in acetone, exposure on ear), 4 days exposure, sacrifice 6 days after final exposure Remark: Hypersensitivity study results not listed here NO(A)EL (mg/kg bw/d) 12.5 LO(A)EL (mg/kg bw/d) 25 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) system, thyroid, endocrine system PFOA Doses: 0, 0.25, 2.5, 6.25, 12.5, 25, 50 mg/kg bw/d 14-day RDT study Rat (SD) n/sex/group = 3 (m only) Exposure: oral (dietary) Liver: Rel. liver weight ( +45%) CAT activity ( +57%) Induction of 80K-protein Remark: Acute toxicity dose response for PFOA also available (i.p. injection, 5-150 mg/kg) Body weight, kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Ikeda et al., 1985) PFOA (ammonium salt, 10% aqueous solution) Doses: 0, 0.02% (equivalent to 20 mg/kg bw/d) 14-day RDT study Rat (Crj:CD(SD)IGS) n/sex/group = 4 (m only) Exposure: oral (gavage) Body weight (increase but ns) Liver: Rel. liver weight ( +102% at LOEL) 5 Cyanide-insensitive palmitoyl CoA -oxidation activity ( +598% 5 at LOEL) Carnitine acetyltransferase activity ( +1960% at LOEL) 5 kidney, nervous system, immune 50 system, 50 haematopoietic system, thyroid, 50 endocrine system (Iwai and Yamashita, 2006) PFOA Doses: 0, 0.5, 5, 50 mg/kg bw/d 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 -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) 43 kidney, nervous system, immune 2.5 5 system, 2.5 5 haematopoietic - 2.5 system, thyroid, 2.5 5 endocrine system - 2.5 - 2.5 5 10 5 10 (Kawashim a et al., 1995) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 0.04 % (equivalent to 2.5, 5, 10, 21, and 43 mg/kg bw/d) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) PFOA (ammonium salt, 99%) 21-day RDT studies Mouse (Crl:CD-1) 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 0.13/0.13 0.13/0.13 380 38 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, 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 14-day RDT study Body weight (-14% at LOEL) 2 20 kidney, nervous (Liu et al., (>99.9%) Rat (Crl:CD BR(CD)) Liver: Abs. + rel. liver weight ( +34% at LOEL) 0.2 2 system, immune system, 1996) n/sex/group=15 (males only) Protein of hepatic microsomes ( +7% at LOEL) Hepatic aromatase activity ( +300% at LOEL) - 0.2 0.2 2 haematopoietic system, thyroid Total hepatic aromatase ( +560% at LOEL) 0.2 2 Exposure: oral (gavage) Hepatic -oxidation activity ( +190% at LOEL) Total hepatic cytochrome P450 ( +70% at LOEL) 0.2 2 2 20 Endocrine system: Doses: 0, 0, 0.02, 2, 20, 40 mg/kg bw/d Serum estradiol ( +56% at LOEL) 0.2 2 (controls ad libitum and pair-fed to 40 Reproductive tissue: mg control) Rel. testes weight ( +12% at LOEL) (compared to ad libitum 2 20 control); ( -11% at 40 mg compared to pair-fed control) PFOA 4-week RDT study Body weight (WT) ( -14% at LOEL) 10.8 21.6 kidney, nervous PFOA concentrations in whole (Minata et (ammonium Mouse (Wild-type mice (129S4/SvlmJ) Body weight gain (WT) ( -167% at LOEL) 5.4 10.8 system, immune blood (WT) (g/ml): al., 2010) salt, >98%) and Ppar-null mice) (129S4/SvJae- Body weight (PPAR KO) ( +11% at LOEL, ns at highest dose) - 5.4 system, 12.5 mol/kg: 20.6 2.4 Ppartm1Gonz/J) Liver: haematopoietic 25 mol/kg: 46.9 3.2 Abs. + rel. liver weight (WT) ( +240% at LOEL) - 5.4 system, thyroid, 50mol/kg:64.2 6.5 Abs./rel. liver weight (PPAR KO) ( +180% at LOEL) - 5.4 endocrine system n/sex/group = 10 (males only) Clinical Chemistry: PFOA concentration in bile Exposure: oral (gavage) Plasma AST (WT) ( +83% at LOEL) 5.4 10.8 (WT) (g/ml): Plasma ALT (WT) ( +577% at LOEL) - 5.4 12.5 mol/kg: 56.8 26.9 Plasma total bilirubin (WT)( -44% at 12.5 mol/kg; +67% at 50 25 mol/kg: 784.0 137.6 44 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 12.5, 25, 50 mol/kg/d (0, mol/kg) 5.4, 10.8, 21.6 mg/kg bw/d) Plasma total cholesterol (WT) ( -17% at LOEL) Plasma triglycerides (WT) ( +47% at LOEL, ns at highest dose) Plasma AST (PPAR KO) ( +535% at LOEL) Plasma ALT (PPAR KO) ( +491% at LOEL) Plasma total bilirubin (PPAR KO)( +683% at 50 mol/kg) Plasma total bile acid (PPAR KO) ( +1350% at 50 mol/kg) Plasma total cholesterol (PPAR KO) ( -38%/-36% at 12.5/25 mol/kg; +66% at 50 mol/kg) Plasma triglycerides (PPAR KO) ( +102% at LOEL) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) 5.4 10.8 - 5.4 10.8 21.6 - 5.4 - 5.4 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 50mol/kg: 2174.0 332.4 PFOA concentration in liver (WT) (g/ml): 12.5 mol/kg: 181.2 6.3 25 mol/kg: 198.8 15.4 50mol/kg: 211.6 13.3 PFOA concentrations in whole blood (Ppar(-/-)) (g/ml): 12.5 mol/kg: 19.3 2.2 25 mol/kg: 36.4 2.7 50mol/kg: 71.2 8.0 PFOA concentrations in bile (Ppar(-/-)) (g/ml): 12.5 mol/kg: 19.6 2.2 25 mol/kg: 62.9 16.7 50mol/kg: 383.0 109.9 PFOA concentrations in liver (Ppar(-/-)) (g/ml): 12.5 mol/kg: 172.3 8.9 25 mol/kg: 218.3 14.5 50mol/kg: 239.7 25.0 PFOA 7-day RDT study (ammonium Rat (Crl:CD(SD)BR) salt, 70:30 linear:branch ed) n/sex/group = 6 (males only) 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 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Pastoor et al., 1987) 45 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks 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) salt, 98% weeks compared to ad libitum, ns compared to pair-fed control) Body weight gain over weeks 1-13 ( -14% compared to ad 1.94 purity) n/sex/group = 5-15 (m only) libitum control, ns compared to pair-fed control) Liver: Rel. liver weight ( +13% at LOEL after 4 weeks, after 7 and 13 0.06 Exposure: oral (dietary) weeks, LOEL at 30 ppm!, after 13 weeks significant reduction of -8% 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) LO(A)EL (mg/kg bw/d) 6.5 6.5 0.64 0.64 0.64 Remark: Effects are reversible in recovery (Serum levels of PFOA decline rapidly in recovery). Overall, "study no effect level was 1 ppm (0.06 g/mg) with doses of 10 ppm (0.64 g/mg) and higher producing adaptive and reversible liver changes." Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) kidney, nervous system, immune system, haematopoietic system, thyroid, clinical chemistry Serum PFOA concentrations after 4 weeks: 0 ppm: 0 (pair fed): 1 ppm: 6.5 1 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: (Perkins et al., 2004) 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 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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) 2/5/10 days Body weight ( -6% at LOEL after 5 days) - Mouse (C57B1/6) Liver: Abs./rel. liver weight ( +75%/+112% at LOEL after 5 days) - n/sex/group = 3 (m only) Hepatic protein content ( +36% at LOEL after 5 days) - Hepatic mitochondrial protein content ( +767% at LOEL after 5 - days) Exposure: oral (dietary) Hepatic microsomal protein content ( +55% at LOEL after 5 24 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 - (0.001, 0.003, 0.01% only for hepatic enzymes) after 5 days) Hepatic cyanide-insensitive palmitoyl-CoA oxidation specific activity - ( +312% at LOEL after 10 days) 24 kidney, nervous system, immune 24 system, 24 haematopoietic 24 system, thyroid, endocrine system 120 24 24 24 1.2 (Permadi et al., 1993) PFOA (98% purity) 10-day study Mouse (C57B1/6) n/sex/group = 3 (m only) Exposure: oral (dietary) Doses: 0, 0.02, (0.1)% (control only for 10 days, 0.1% only 2 and 5 days) (equivalent to 24, 120 mg/kg bw/d) 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 6) Microsomal cytochrome P450 reductase ( +100% in Table5, +285% in Table 6) Microsomal Cytochrome B5 () (ns in Table 5, +70% in Table 6) Microsomal epoxide hydrolase ( +239% in Table5, +191% in Table 6) Hepatic cytosolic DT-diaphorase ( +391% in Table7, +438% in Table 8) 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 47 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Permadi et al., 1992) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks consumption in Fe presence) ( -54%/-55% in Exp.1/Exp.2) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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... PFOA (96% 10-day study Body weight (data not shown) purity) Mouse (C57BL/6 (H-2b)) Liver weight (data not shown) Immune system: n/sex/group = 4 (m only) Number of total white blood cells ( -72%) Number of lymphocytes ( -73%) 0.34 4.3 0.34 4.3 Number of neutrophils ( -52%) 0.34 4.3 Exposure: oral (dietary) LPS-induced TNF- production (Ex vivo, in vitro LPS-stimulation) 0.34 4.3 ( +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 4.3 (equivalent to 0, 0.34, 4.3 mg/kg bw/d) in peritoneal cavity, +50% in bone marrow, +152% in spleen) LPS-induced TNF- production (Ex vivo, in vivo LPS-stimulation) 0.34 4.3 (+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 4.3 in peritoneal cavity, +150% in bone marrow); ( -60% in spleen) Serum TNF- levels after LPS injection ( +473%) 0.34 4.3 Serum IL-6 levels without LPS injection ( +150%) 0.34 4.3 Serum IL-6 levels with LPS injection ( +92%) 0.34 4.3 PFOA (96% purity) 10-day-study Mouse (C57BL/6 (H-2b)) n/sex/group = 4-7 (m only) Exposure: oral (dietary) Dose: 0, 0.002% (w/w) (equivalent to 0.65 mg/kg bw/d) Body weight gain (ns) Liver: Rel. liver weight ( +67%) Histological alterations in structure of parenchymal cells Hypertrophy of hepatocytes around central vein Larger cell surface in centrilobular hepatocytes Total number of intrahepatic immune cells ( +100%) Total number of intrahepatic myeloid-, lymphoid-, and erythroidrelated cells (e.g. +64% for granulocytes, +118% for macrophages, +267% for myeloid suppressor cells, +283% for 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 system, haematopoietic system, thyroid, endocrine system, clinical chemistry Serum PFOA concentration after 10 days: (Qazi et al., 2009) 0.02%: 152 8.6 g/ml (357 20.8 M) kidney, nervous Serum PFOA concentration system, after 10 days: haematopoietic 0.002%: 87.6 2.1 g/ml system, thyroid, (211.6 5.1 M) endocrine system (Qazi et al., 2010) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFOA (96% 10-day study (+10 days recovery) Body weight ( -27% at LOEL) purity) Mouse (C57BL/6 (H-2b)) Liver: Rel. liver weight ( +73% at LOEL) n/sex/group = 8 (m only) Immune system: Rel. thymus weight ( -79% at LOEL) Rel. spleen weight ( -44% at LOEL) Exposure: oral (dietary) 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) 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. Pair- fed 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 NO(A)EL (mg/kg bw/d) 0.75 LO(A)EL (mg/kg bw/d) 7.5 - 0.4 0.75 7.5 0.75 7.5 0.75 7.5 0.75 7.5 0.4 0.75 Key parameters / targets not addressed kidney, nervous system, haematopoietic system, thyroid, endocrine system, clinical chemistry Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) (Qazi et al., 2012) PFOA (no 5-week Body weight (ns) purity Mouse (C57BL/6 and BALB/c, specified, but premature) supplier was Liver: Rel. liver weight (C57) (m/f, +67%/+66%) Rel. liver weight (BALB/c) (m/f, +55%/+65%) Sigma Aldrich which today n/sex/group = 2x3 Liver cholesterol (BALB/c) (m/f, -80/60% in BALB/c mice) PPAR 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) kidney, nervous system, haematopoietic system, thyroid PFOA levels in plasma in C57 male/female mice: Control: 2/28 ng/mL PFOA exposed: 26.9/44.3 g/mL PFOA levels in plasma in BALB/c mice: Control: 5/86 ng/mL PFOA-exposed: 28.2/35.6 g/mL (Rebholz et al., 2016) PFOA (ammonium salt, 98% purity) 21-day study Mouse (ICR) 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- gene ex ( -50% at LOEL) Hepatic IL-1 gene ex ( -60% at LOEL) Hepatic TGF- gene ex ( +40% at LOEL) Immune system: 2.64 2.64 0.49 2.64 2.64 17.63 2.64 17.63 17.63 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) 49 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 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) Alterations of thymic T-lymphocyte populations ( in CD4-CD8-, CD4-CD8+; in CD4+CD8+) Splenic TNF- gene ex ( +83% at LOEL) Splenic IL-1 gene ex ( +100% at LOEL) Splenic TGF- gene ex ( +100% at LOEL) Splenic c-myc gene ex ( +150% at LOEL) Thymic c-myc gene ex ( +90% at LOEL) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed - 0.49 17.63 2.64 17.63 17.63 2.64 47.21 17.63 47.21 47.21 17.63 Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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 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) Kidney, nervous system, thyroid, endocrine system (Tan et al., 2013) PFOA (96%) 7-day-study Mouse (Balb/c) n/sex/group = 3x5 (f) 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 control -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, haematopoietic system, thyroid, endocrine (Vetvicka and Vetvickova, 2013) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Immune system: Exposure: oral (gavage, in PBS) Rel. spleen and thymus weight (ns) Thymic cellularity ( -47/-21% without/with RVB 300) Doses: 0, 20 mg/kg bw/d 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) NO(A)EL (mg/kg bw/d) LO(A)EL (mg/kg bw/d) Key parameters / targets not addressed system, clinical chemistry Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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 was not specified, but Sigma Aldrich delivers in 95% purity) 7-day-study+2, 5, or 10 days recovery Mouse (C57BL/6) n/sex/group = 4 (m) Exposure: oral (dietary) Doses:0, 0.02% (w/w) (equivalent to 24 mg/kg bw/d) Body weight ( -20%, recovery after 5 days) Liver Abs liver weight ( +90%, further increase during recovery: +110% after 2 days, +130% after 5 days recovery, slow recovery after 10 days (+80%)) Hepatic CPT activity ( +300%, slow recovery (down to +160% after 10 days)) Hepatic COT activity ( +900%, slow recovery (down to +400% after 10 days) Fat tissue 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) PFOA 10-day-study, mouse (C57BL/6) n/sex/group = 4 (m) Exposure: oral (dietary) Body weight ( -17%) Liver Abs./rel. liver weight ( +96%/+136) Immune system: Abs. + rel. thymus weight ( -85%) Abs.+ rel. thymic DNA contet ( -39% for rel) Abs. + rel. spleen weight ( -30%) Abs. splenic DNA content ( -49%) 51 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) 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) Doses: 0, 0.02% (w/w) (equivalent to Thymocyte cellularity ( -56% for CD4-CD8-, -95% for CD4+CD8+, 30 mg/kg bw/d) -64% for CD4+, -72% for CD8+) Splenocyte cellularity ( -75% for CD3+, -78% for CD4+, -74% for CD8+, -86% for CD19+) LO(A)EL (mg/kg bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) PFOA PFOA PFOA Remark: most effects already present after 5 days 10-day-study, 2-10 days Body weight ( -15% at 0.02% after 5 days, data not shown for Kidney, nervous Mouse (C57BL/6) other doses) Liver: system, haematopoietic n/sex/group = 4 (m) Rel. liver weight ( +60% at LOEL) Immune system: 11.5 23 system, thyroid, endocrine Rel. thymus weight ( -50% at LOEL) 3.5 11.5 system, clinical Exposure: oral (dietary) Rel. spleen weight ( -35% at LOEL) 3.5 11.5 chemistry Hepatic acyl-CoA oxidase activity ( +300% with palmitoyl-CoA as - 1 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) 7-day-study Body weight ( -14% in WT) kidney, nervous Mouse (C57BL/6 (WT) or PPAR-null) Liver: Abs. liver weight ( +86%/+119% in WT/PPAR-null) system, haematopoietic n/sex/group = 4 (m) Immune system: Abs. spleen weight ( -39% in WT) system, immune system, Splenocyte number ( -78% in WT) endocrine Exposure: oral (dietary) Abs. thymus weight ( -79%/-39% in WT/ PPAR-null) Thymocyte number ( -84%/-39% in WT/ PPAR-null) system, clinical chemistry 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+CD8- 33.3 mg/kg bw/d (WT) or 29.8 mg/kg , -68% for CD4-CD8+, -55% for CD4-CD8-) bw/d (PPAR)) Splenocyte cellularity ( -42% for CD3+, -85% for CD19+) 10-day-study, 2-10 days Mouse (C57BL/6) n/sex/group = 4 (m) Exposure: oral (dietary) Immune system: IgM ( -73%/-72% without/with HRBC- immunisation) IgG1 ( -29%/-99.95% without/with HRBC-immunisation) IgG2b ( -50%/-95% without/with HRBC-immunisation) IgG3 ( -33%/-91% without/with HRBC-immunisation) Remark: IgM only antibody almost recovered after 6 days, other Body weight, liver, kidney, nervous system, haematopoietic system, endocrine system, clinical 52 (Yang et al., 2001) (Yang et al., 2002b) (Yang et al., 2002a) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks antibodies: not fully recovered NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) chemistry Doses: 0, 0.02% (w/w) (equivalent to 30 mg/kg bw/d) PFOA 14 days-study + 14 days recovery Body weight decreased in 14 days of treatment (compared to intial (ammonium Rabbit (albino) salt) 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 Rat (Crl:CD) n/sex/group = 15 (m) Exposure: Dermal (skin patch on back, 15% of body surface), 6h/day (10 applications: 5 application days, 2 rest days, 5 application days) Doses: 0, 20, 200, 2000 mg/kg Body weight (day 5) ( -13%) Body weight (day 10) ( -15%) 20 Body weight (recovery day 14) ( -8%) 20 Liver 20 Abs./rel. liver weight directly after last application ( +53%/+55% at LOEL) 0 Abs./rel. liver weight after 14 days of recovery ( +33%/+54% at LOEL) 20 Abs./rel. liver weight after 42 days of recovery ( +30%/+31% at LOEL) 200 Haematopoietic system: Red blood cell directly after last application ( +53%/+55% at LOEL) 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 53 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 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 2000 mg/kg:8.2 4.1 PFOA (ammonium salt, 100%) Repeated-dose-inhalation Rat (Crl:CD) n/sex/group = 24 (m) Exposure: inhalation, 6h/day (10 applications: 5 application days, 2 rest days, 5 application days) Doses: 0, 1, 8, 84 mg/m 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% at LOEL) 1 Abs./rel. liver weight after 14 days of recovery ( +19%/+23% at LOEL) 1/8 Abs./rel. liver weight after 28 days of recovery ( +16%/+23% at LOEL) 1/8 Clinical chemistry: Serum ALP activity (after last application) ( +36% at LOEL) 1 Serum ALP activity (after 14 days recovery) ( +38% at LOEL) 8 84 8 8/84 8/84 8 84 APFO concentrations in blood (ppm) after last application: 0 mg/m: 1.4 1 mg/m: 13 8 mg/m: 47 84 mg/m: 108 (Kennedy et al., 1986) Organofluorine concentrations in blood (ppm) after 14 days recovery: 0 mg/m: 0.28 1 mg/m: NA 8 mg/m: NA 84 mg/m: 10 Organofluorine concentrations in blood (ppm) after 28 days recovery: 0 mg/m: 0.10 1 mg/m: 1.2 8 mg/m: 3.8 84 mg/m: 7.1 Organofluorine concentrations in blood (ppm) after 42 days recovery: 0 mg/m: 0.032 1 mg/m: NA 8 mg/m: NA 84 mg/m: 1.8 Organofluorine concentrations in blood (ppm) after 84 days recovery: 0 mg/m: 0.015 1 mg/m: NA 8 mg/m: NA 84 mg/m: 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) 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 54 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) Rat (SD) Hepatocyte hypertrophy -/50 Acetyl-CoA activity (m, +222% at LOAEL) - n/sex/group = 10 (m+f) Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 ( +246- 1971% at LOAEL) - Kidney: Exposure: oral (gavage, in distilled Abs./rel. kidney weight (m/f, +11/+7% at LOAEL) -/25 water) Immune system: Abs. spleen weight (m, -12% at LOAEL; f: ns) 1.25 Doses (m): 0, 0.625, 1.25, 2.5, 5, 10 mg/kg bw/d Abs. thymus weight (m, -12% at LOAEL; f: ns) Thyroid: Rel. thyroid weight (m, +17% at LOAEL; f: ns)) 5 0.625 Doses (f): 0, 6.25, 12.5, 25, 50, 100 Thyroid gland follicular cell hypertrophy 5/50 mg/kg bw/d Haematopoietic system: Erythrocytes , haemoglobin, Haematocrit (m, -4-6% at LOAEL) 0.625 Reticulocyte (m, -15% at LOAEL) - Clinical chemistry: ALT (m, +19-47%) - ALP (m, +13-89%) - ALP (m, +22-38%) 1,25 Cholesterol (m, -37%) - Endocrine system (m): T3 ( -40% at LOAEL), free T4 ( -79% at LOAEL), total T4 ( -91% at LOAEL) -/50 Reproductive Tissue (m only): L. Cauda Epididymis weight (m, -11% at LOAEL) 2.5 L. Epididymis weight (m, -9% at LOAEL) 5 Sperm (106/g cauda epididymis) (m, -24% at LOAEL) 5 Other effects: Hyperplasia/inflammation of respiratory epithelium -/6.25 LO(A)EL (mg/kg bw/d) 0.625/100 0.625 0.625 Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) higher plasma concentration compared to females across the dose groups (up to 1,000fold higher levels) 0.625/50 2.5 10 1.25 10/100 1.25 0.625 0.625 0.625 2.5 0.625 0.625/100 5 10 10 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) PFNA, 5-day RDT study, non-guideline study PerfluorononaRat (Wistar) noic acid (analytical grade), EC: n/sex/group = 4 206-801-3, CAS: 375-95- Exposure: i.p. injection (vehicle: 1, C9HF17O2 propyleneglycol:water (1:1, v:v) Liver: Peroxisomal -oxidation (m/f) -/5 Remarks: Mechanistic study to assess the MoA; peroxisomal oxidation is statistically highly correlated with PFCA concentration in the liver (r=0.850, p<0.001) internal dose in liver decisive of effect not carbon chain length or sex 2.5/10 kidney, nervous PFNA conc. in the liver at system, immune highest dose: system, haematopoietic system, thyroid, m: 358 19 g/g liver f: 102 11 g/g liver endocrine system (Kudo et al., 2000) 55 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) PFCAs assessed: PFHpA, PFOA, PFNA, PFDA PFNA 5-day RDT study, non-guideline study Liver: Mouse (ddy) Abs./rel. liver weight -/- Peroxisomal -oxidation (m/f) -/- n/sex/group = 3-5 Activity of hepatic microsomal 1-Acly-GPC acyltransferase (m, only - 20 mg/kg bw/d tested) Exposure: i.p. injection (vehicle: propyleneglycol:water (1:1, v:v) Remarks: Mechanistic study to assess the MoA; hepatic peroxisomal -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) internal dose in liver decisive for effect Doses: 0, 2.5, 5, 10, 15, 20 mg/kg bw/d 2.5/2.5 2.5/2.5 20 kidney, nervous C-length and sex dependent (Kudo et system, immune accumulation of PFCAs in the al., 2006) system, liver (higher concentrations in haematopoietic males and with higher C system, thyroid, length) endocrine system 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: Hepatocytes with focal vacuolations mRNA levels of CYP4A1 and ACOX Hepatic TNF level Lipid accumulation in liver Remarks: Mechanistic study to assess the MoA kidney, nervous 1 5 system, immune - 0.2 system, - 0.2 haematopoietic 1 5 system, thyroid, endocrine system (Fang et al., 2012b) PFNA (97%) Doses: 0, 0.2, 1, 5 mg/kg bw/d 14-days RDT study, non-guideline Rat (SD) n/sex/group = 6 (m) Exposure: oral (gavage, in distilled water) Liver: MDA in liver Glycogen in liver mRNA of G6PC/GLUT2 Clinical chemistry: Serum glucose ( +9% at LOEL) Serum HDL ( -18-55%) Serum LDL ( +100% at LOEL) kidney, nervous 1 5 system, immune 1 5 system, 1 5 haematopoietic system, thyroid, 0.2 1 endocrine system - 0.2 1 5 Doses: 0, 0.2, 1, 5 mg/kg bw/d 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 (Fang et al., 2012a) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks in the rat liver PFNA (97%) 7-days RDT study, non-guideline Liver: Rat (SD, diabetes induced via streptozotocin) Hepatic cholesterol ( +91% at LOEL) Hepatic triglycerides ( +180% at LOEL) G6P-dehydrogenase activity ( +19% at LOEL) n/sex/group = 10 (m) Hepatic lipase ( -68% at LOEL) Clinical chemistry: Serum ALT ( +60% at LOEL) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 0.2 1 1 5 0.2 1 1 5 0.2 1 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Fang et al., 2015) Exposure: oral (gavage, in distilled water) 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 Body weight ( -15% at LOEL) 1 3 Liver, kidney, Mouse (BALB/c (SD)) Body weight gain ( -517%) nervous system, Immune system: Clinical chemistry n/sex/group = 6 (m) Abs. spleen weight ( -9.6% (text), -34% (graph) at LOEL) 1 3 Rel. spleen weight ( -32%) 3 5 Splenocyte apoptosis ( +90%) 3 5 Exposure: oral (gavage) Abs./rel. thymus weight ( -33.3%/-39% at LOEL) Thymocyte apoptosis ( -+67%) 1 3 3 5 Doses: 0, 1, 3, 5 mg/kg bw/d IL-4 in spleen homogenates ( -17%) IFN- in spleen homogenates ( -50%) - 1 3 5 Thymic gene expression of PPAR- ( +510%/6.1-fold at 1 mg/kg) Thymic gene expression of PPAR- ( +160%/2.6-fold at 1 mg/kg) Thymic gene expression of IL-1- ( +200%/3-fold at 1 mg/kg) Endocrine system: ACTH ( +72% at LOEL) 3 5 Cortisol ( +17% at LOEL) 1 3 PFNA (97%) 14-day-study Body weight ( -18% at LOEL) 1 3 Liver, kidney, Rat (SD) Immune system: Abs. thymus weight ( -20% at LOEL) 1 3 nervous system, Clinical chemistry n/sex/group =6 (m) Rel. thymus weight ( -58% at LOEL) Thymocyte apoptosis 3 5 Serum IL-1 ( +215% at LOEL) 1 3 Exposure: Oral (gavage) Serum IL-2 ( -29% at LOEL) Serum IL-4 ( +106% at LOEL) 1 3 3 5 Thymic PPAR gene ex. ( +120% at LOEL) - 1 Doses: 0, 1, 3, 5 mg/kg bw/d Thymic PPAR gene ex. ( +190% at LOEL) 1 3 Endocrine system: 57 (Fang et al., 2008) (Fang et al., 2009) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Cortisol ( +67% at LOEL) NO(A)EL (mg/kg bw/d) 3 LO(A)EL (mg/kg bw/d) 5 Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) PFNA (97%) 14-day-study Rat (SD) n/sex/group = 6 (m) Exposure: oral (gavage) Doses: 0, 1, 3, 5 mg/kg bw/d Remark: More data (mainly related to MAPK and NF-B signalling pathways) available Feng 2009, 2010 and Fang 2009, 2010 probably all one experiment and different endpoints published separately Immune system Body weight, Abs. spleen weight ( -22% at LOEL) - 1 liver, kidney, Rel. spleen weight ( -8% at LOEL) 3 5 nervous system, Lymphoid cell apoptosis in spleen 1 3 endocrine Splenic IL-1 ( +49% at LOEL) 3 5 system, clinical Splenic IL-6 ( +51% at LOEL) 3 5 chemistry Splenic TNF- ( +30% at LOEL) 3 5 Splenic IFN ( -35% at LOEL) 3 5 Splenic IL-10 ( -57% at LOEL) 3 5 Splenic H2O2 conc. ( +31% at LOEL) 3 5 Splenic SOD activity ( -42% at LOEL) 1 3 Splenic PPAR gene ex. ( +160% at LOEL) 1 3 Splenic PPAR gene ex. ( +130% at LOEL) 1 3 (Fang et al., 2010) PFNA (97%) 14-day-study Rat (SD) n/sex/group =6 (m) Exposure: oral (gavage) Remark: IL-1, IL-6, IFN, and TNF- are pro-inflammatory, IL-10 is anti-inflammatory. More protein expression data (by Western blotting) available Endocrine system Serum testosterone ( -85% at LOEL) 3 5 Serum estradiol ( +104% at LOEL) 3 5 Apoptotic cells in testes ( +367% at LOEL) 1 3 Fas gene ex ( +90% at LOEL) 3 5 Bax gene ex ( +36% at LOEL) 3 5 Bcl-2 gene ex ( -27% at LOEL) 1 3 (Feng et al., 2009) PFNA (97%) Doses: 0, 1, 3, 5 mg/kg bw/d 14-day-study Rat (SD) n/sex/group =6 (m) Exposure: oral (gavage) 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) 58 1 3 - 1 - 1 3 5 - 1 (Feng et al., 2010) 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 Observed effects NO(A)EL (%), EC/CAS, exposure, doses (guideline/similar (mg/kg formula to guideline/non-guideline) Remarks bw/d) Remark: in vitro study with primary sertoli cells also available. Doses: 0, 1, 3, 5 mg/kg bw/d Overall, the study shows that PFNA treatment led to the damage of specific secretory functions of Sertoli cells PFNA 14-day RDT studies Mortality (all mice at 300 and 3000 ppm died before end of study) 3.8 (ammonium Mouse (Crl:CD-1) Body weight (data not shown) 1.3 salt, 99%) Liver n/sex/group = 5 Abs. liver weight (m/f, +58/+56% at LOEL) -/- Rel. liver weight (m/f, +48/+35% at LOEL) -/- LO(A)EL (mg/kg bw/d) 38 3.8 0.38/0.38 0.38/0.38 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Kennedy, 1987) Exposure: oral (dietary) PFNA (97%) Doses: 0, 3, 10, 30, 300, 3000 ppm (equivalent to 0, 0.38, 1.3, 3.8, 38, 380 mg/kg bw/d) 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 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) PFNA (purity 14-days RDT study, non-guideline not specified) study Rat (Wistar) n/sex/group = 10 (m) Remarks Mechanistic study to assess the MoA Body weight ( -31% at LOAEL) Liver: Hepatic OATP4C1 protein Clinical chemistry: Serum corticosterone 1 5 kidney, nervous system, immune - 0.2 system, - 0.2 haematopoietic - 0.2 system, thyroid, - 0.2 endocrine system 1 5 1 5 1 5 1 5 (Wang et al., 2015) 0.25 5 kidney, nervous Serum levels (g/ml): (Hadrup et system, immune at 0.0125 mg/kg bw/d: 0.396 al., 2016) - 0.0125 system, haematopoietic at 0.25 mg/kg bw/d: 30 - 0.0125 system, thyroid, at 5 mg/kg bw/d: 602 endocrine system Exposure: oral (gavage) Doses: 0, 0.0125, 0.25, 5 mg/kg bw/d 59 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFNA (purity 3-day RDT study, non-guideline study Liver: not specified) Mouse (C57BL/6) Rel. liver weight ( +88%) mRNA of Cyp4A10 and Cyp2b10 n/sex/group = 4 Remarks Mechanistic study to assess the MoA NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) - 20 - 20 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) kidney, nervous (Abe et al., system, immune 2017) system, haematopoietic system, thyroid, endocrine system Exposure: i.p. (vehicle: corn oil) Doses: 0 , 20 mg/kg bw/d PFNA 7-day RDT study, non-guideline study Liver: kidney, nervous Mouse (SV129) Abs. + rel. liver weight ( +120%) Hepatic triglyceride ( +260%) - 10 system, immune - 10 system, n/sex/group = 4 (m) Hepatic lipid content ( +540%) - 10 haematopoietic system, thyroid, Remarks endocrine system Exposure: oral (gavage) Mechanistic study to assess the MoA of PFOA, PFNA, PFHxS (Das et al., 2017) PFNA PFNA (>98%) Doses: 0 , 10 mg/kg bw/d 7-day RDT study, non-guideline study Liver: Mouse (129S1/SvlmJ) Abs. + rel. liver weight ( +56%) - Remarks n/sex/group = 4 (m) Mechanistic study to assess the MoA of PFOA, PFNA, PFHxS, PFOS: gene expression profiles significantly similar to profiles from mouse tissues exposed to agonists of the constitutive activated receptor Exposure: oral (gavage) (CAR), estrogen receptor (ER), and PPAR; PPAR and ER were activated by all four PFCAs in trans-activation assays from the ToxCast screening program Doses: 0 , 1, 3 mg/kg bw/d 28-day RDT study, NTP study, similar to Mortality (m/f, 80-90% at LOAEL) 2.5/6.25 OECD TG 407 Body weight (m/f, -17/-10% at LOAEL) 0.625/ Rat (SD) Liver: 1.56 Abs. + rel. liver weight (m/f, +23/+21% at LOAEL) - n/sex/group = 10 Hepatocyte, hypertrophy (m, 70% incidence at LOAEL) -/3.12 Hepatocyte, cytoplasmic alteration (m, 100% incidence at LOAEL) -/- Hepatocyte necrosis (m, 50% incidence at LOAEL) 1.25/6.25 Exposure: oral (gavage, in distilled water) Hepatocyte, cytoplasmic vacuolization (m, 60% incidence at LOAEL) 0.625 Acetyl-CoA activity (m, +391% at LOAEL) - Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 ( +216- - kidney, nervous 1 system, immune system, haematopoietic system, thyroid, endocrine system (Rosen et al., 2017) 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 60 Commented [A22]: How important is it to fill in this? Most relevant for guideline studies deviating form guideline? Formatted: French (France) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 1889% at LOAEL) 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 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: NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) -/- 0.625/1.56 0.625/1.56 0.625/3.12 0.625 0.625 1.25/6.25 2.5/6.25 1.25/6.25 2.5/6.25 2.5/6.25 1.25/3.12 1.25/6.25 1.25 1.25 2.5/12.5 5/12.5 2.5/12.5 5/12.5 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 (g/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 (g/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 (g/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 61 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Olfactory Epithelium Degeneration in f Forestomach: Epithelium hyperplasia, chronic inflammation NO(A)EL (mg/kg bw/d) 12.5 1.25/6.25 LO(A)EL (mg/kg bw/d) 25 2.5/12.5 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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) PFDA, 7-day RDT study, non-guideline study Perfluorodeca Rat (Wistar) noic acid (purity not specified), EC: n/sex/group = 4 (m) 206-400-3, CAS: 335-76- Exposure: oral (diet) 2, C10HF19O2 Doses: 0, 0.00125, 0.0025, 0.005 or 0.01% (equivalent to 0 , 1.5, 3, 6, 12 mg/kg bw/d) Body weight Liver: Abs./rel. liver weight Peroxisomal -oxidation Microsomal I-acyl-GPC acyltransferase Cytosolic long-chain acyl-CoA hydrolase GSH S-transferase Hepatic Triacylglycerol Hepatic Cholesterol 6 12 - 1.5 1.5 3 - 1.5 1.5 3 3 6 - 1.5 6 12 PFDA 5-day RDT study, non-guideline study Liver: (analytical grade) Rat (Wistar) Peroxisomal -oxidation (m/f) 2.5/2.5 5/5 n/sex/group = 4 Remarks Mechanistic study to assess the MoA; peroxisomal -oxidation is statistically highly correlated with PFCA concentration in the liver Exposure: i.p. injection (vehicle: propyleneglycol:water (1:1, v:v) (r=0.850, p<0.001) internal dose in liver decisive of effect not carbon chain length or sex kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Kawashim a et al., 1995) kidney, nervous PFDA conc. in the liver at system, immune highest dose: system, haematopoietic system, thyroid, m: 453 19 g/g liver f: 412 33 g/g liver endocrine system (Kudo et al., 2000) 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 not specified) Mouse (C57BL/6) Liver: Rel. liver weight ( +55%) mRNA of Cyp4A10 n/sex/group = 4 (sex not reported) Exposure: i.p. (vehicle: corn oil) Remarks Mechanistic study to assess the MoA kidney, nervous - 20 system, immune - 20 system, haematopoietic system, thyroid, endocrine system (Abe et al., 2017) Doses: 0, 20 mg/kg bw/d PFDA (97.3%) 28-day RDT study, NTP study to assess Body weight ( -22%) 1 2 nervous system, 62 (Frawley et 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks immunotoxicity and hepatotoxicity Liver: Rat (SD) Abs. liver weight Rel. liver weight n/sex/group = 8 (f) Single cell hepatocyte necrosis (minimal severity) Kidney: Abs. kidney weight Exposure: oral (gavage) Rel. kidney weight Haematopoietic system: Mean corpuscular haemoglobin ( -6% at LOAEL) Doses: 0, 0.125, 0.25, 0.5, 1, 2 mg/kg bw/d NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) 0.125 - 0.25 0.25 0.125 0.5 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) thyroid, al., 2018) endocrine system 0.25 0.5 0.125 0.25 0.125 0.25 PFDA 28-day RDT study, NTP study to assess immunotoxicity and hepatotoxicity Mouse (B6C3F1/N) n/sex/group = 8 (f) Exposure: oral (gavage) Doses: 0, 0.31, 0.625, 1.25, 2.5, 5 mg/kg bw/week LOAEL/NOAELs given in mg/kg bw/week Liver: Abs./rel. liver weight Immune system: Rel. spleen weight Total spleen cell numbers ( -24% at LOAEL) Number of B-cells ( -27% at LOAEL) Number of T-cells ( -22% at LOAEL) Number of T-helper cells ( -19% at LOAEL) Number of cytotoxic T-lymphocytes ( -19% at LOAEL) Number of NK cells ( -18% at LOAEL) Number of macrophages ( -21% at LOAEL) 0.31 0.625 2.5 2.5 0.625 0.625 0.31 0.625 0.31 0.625 1.25 5 5 1.25 1.25 0.625 1.25 0.625 nervous system, thyroid, endocrine system (Frawley et al., 2018) PFDA (98%) 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%) kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Permadi et al., 1992; Permadi et al., 1993) 63 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Hepatic cytosolic epoxide hydrolase ( +144%) PFDA 96% 10 day, non-guideline ReproTox study Gd 10-13: Mouse (C57BL/6N) Maternal body weight gain ( -61% at LOEL) Abs. liver weight ( +10% at LOEL) n/sex/group = 10-14 Rel. liver weight ( +3% at LOEL) Gd 6-15: Exposure: oral (gavage, in corn oil) Maternal body weight gain ( -92% at LOEL) Abs./rel. liver weight ( +61%/+127% at LOEL) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 8 16 0.5 1 0.25 0.5 3 6.4 0.3 1 kidney, nervous system, immune system, haematopoietic system, thyroid, endocrine system (Harris and Birnbaum, 1989) 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) 0.625/0.625 1.25/1.25 OECD TG 407 Liver: Rat (SD) Abs./rel. liver weight ( +11/12% in m/f at LOAEL) -/3.12 0.125/0.125 Hepatocyte, hypertrophy ( 80-100% incidence at LOAEL) 0.625/0.625 1.25/1.25 n/sex/group = 10 Hepatocyte, cytoplasmic alteration (80-100% incidence at LOAEL) 0.325/0.325 0.625/0.625 Hepatocyte necrosis (f only, 40% incidence at LOAEL) 1.25 2.5 Exposure: oral (gavage, in distilled water) Hepatocyte, cytoplasmic vacuolization (90-100% incidence at LOAEL) 0.625/1.25 Acetyl-CoA activity (m only, +22-404%) - 1.25/2.5 0.156 Gene expression of Acox1, Cyp4a1, Cyp2b1, Cyp2b2 -/- 0.156/0.156 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) 0.312/0.156 0.625/0.312 Immune system: Abs. spleen weight (m/f, -26/-36% at LOAEL) 0.625 1.25 Rel. spleen weight (m/f, -19/-9% at LOAEL) 1.25/0.312 2.5/0.625 Abs.thymus weight (m/f, -44/-65% at LOAEL) 0.625/1.25 1.25/2.5 Rel. thymus weight (m/f, -29/-46% at LOAEL) 0.625/1.25 1.25/2.5 Bone marrow hypocellularity (100% incidence at LOAEL) 1.25/1.25 2.5/2.5 Thymus atrophy (80-90% incidence at LOAEL) 1.25/1.25 2.5/2.5 Thyroid: Abs.+rel. thyroid weight (m/f, +27-45%) 0.625/0.156 1.25/0.312 Haematopoietic system (only in m determined): Reticulocytes (-54-62% at LOAEL) 0.625/0.625 1.25/1.25 64 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 (M/mol/kg), values decreased with increasing dose Plasma concentrations after 28-d in m (g/mL): 0.156 mg/kg bw/d: 8.5 0.312 mg/kg bw/d: 23 0.625 mg/kg bw/d: 47.7 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 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 bw/d) bw/d) addressed (time of sampling) Erythrocytes ( +13-23% at LOAEL) 1.25/0.625 2.5/1.25 1.25 mg/kg bw/d: 101.6 Mean cell haemoglobin Endocrine system (m): Adrenal gland weight abs. ( -15% (m) to -36% (f) at LOAEL) Testosterone in m ( -64% at LOAEL) 0.312/1.25 0.625/2.5 -/1.25 0.156/2.5 0.625 1.25 2.5 mg/kg bw/d: 259.4 Liver concentrations after 28-d in m (g/mL): 0.156 mg/kg bw/d: 44.7 Testosterone in f ( +32-355%) free T4 ( -39-42% at LOAEL) T3, female only ( +24% at LOAEL) Clinical chemistry: Globulin (g/dL) (-14-22% at LOAEL) 0.156 0.312 -/1.56 0.625/3.12 0.156/0.625 0.312/1.25 -/0.156 0.156/0.312 0.312 mg/kg bw/d: 87.2 0.625 mg/kg bw/d: 163.9 1.25 mg/kg bw/d: 308.8 2.5 mg/kg bw/d: 403.6 Urea nitrogen ( +25-38% at LOAEL) 0.625/0.625 1.25/1.25 Glucose (-31% at LOAEL) ALT ( +44-45% at LOAEL) ALP ( +22-35% at LOAEL) AST (m, +14-30% at LOAEL) 0.625/0.625 1.25/1.25 0.156/0.625 0.312/1.25 0.156/0.156 0.312/0.312 -/0.625 0.156/1.25 Plasma concentrations after 28-d in f (g/mL): 0.156 mg/kg bw/d: 11.2 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) Bile salts (m, +205-440% at LOAEL) Reproductive Tissue (m only): L. Cauda Epididymis weight (m, -11% at LOAEL) 0.625 1.25 0.625/0.625 1.25/1.25 0.625 1.25 0.625 mg/kg bw/d: 50.3 1.25 mg/kg bw/d: 117.2 2.5 mg/kg bw/d: 246.9 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) PFUnDA 42-day RDT study with the (98.5%), Reproduction/Developmental Toxicity PerfluoroundeScreening Test, according to OECD TG canoic acid, 422 EC: 218-165-4,Rat Crl:CD (SD) CAS: 2058-94- 8, C11HF21O2 n/sex/group = 12 Exposure: oral (gavage, vehicle: corn oil) Liver: Abs. liver weight Rel. liver weight Hepatocyte hypertrophy (58-52% incidence at LOAEL) Immune system: Abs./rel. spleen weight (m only, -19-23% at LOAEL) Clinical chemistry: Serum ALP, ALT (m only, +26% for ALT and +139% for ALP) Serum urea nitrogen ( +46-61% at LOAEL) Serum albumin (m only, -7% at LOAEL) Total protein ( -10-11% at LOAEL) 0.3/0.3 0.1/0.3 0.3/0.3 0.3/0.3 0.3 0.3/0.3 0.3 0.3/0.3 1/1 0.3/1 1/1 1/1 1 1/1 1 1/1 nervous system, endocrine system (Takahashi et al., 2014) 65 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 0.1, 0.3, 1.0 mg/kg bw/d NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters / targets not addressed Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 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, 14-day RDT study PerfluorododeRat (SD) canoic acid (>99%), EC: 206-203-2, n/sex/group = 6 (m) CAS: 307-551, C12HF23O2 Exposure: oral (gavage), vehicle: 0.5% Tween-20) Liver: Abs. liver weight ( -19-26%) Rel. liver weight ( +25-30% at LOAEL) Hepatic triglyceride ( +27% at LOAEL) Hepatic SOD/CAT activity Hepatic mRNA of PPAR/, ACOX, CPT1, Cyp4A1, LDLR Hepatic content of cholesterol ( +39% at LOAEL) Clinical chemistry: Serum triglyceride ( +96% at LOAEL) kidney, nervous 1 5 system, immune 1 5 system, 5 10 haematopoietic - 1 system, thyroid, - 1 endocrine system 5 10 5 10 (Zhang et al., 2008) Doses: 0, 1, 5, 10 mg/kg bw/d PFDoDA 110-day RDT study Body weight ( -7% at LOAEL) 0.2 0.5 kidney, nervous (95%) Rat (SD) Liver: Abs./rel. liver weight ( +10-18%) system, immune - 0.02 system, n/sex/group = 10 (m) Lipid droplets and widespread disintegrated cell systems Swollen and vacuolated hepatocytes 0.02 0.05 0.05 0.2 haematopoietic system, thyroid, Hepatic mRNA of PPAR, CYP4A1, ACOX, Cd36 0.05 0.2 endocrine system Exposure: oral (gavage), vehicle: 0.2% Hepatic triglycerides Tween-20) Clinical chemistry: Serum ALP (m only, +73% at LOAEL) 0.05 0.2 Serum urea nitrogen ( +17% at LOAEL) 0.05 0.2 Doses: 0, 0.02, 0.05, 0.2, 0.5 mg/kg Serum glucose ( +9% at LOAEL) - 0.02 bw/d Serum albumin ( +6% at LOAEL) - 0.02 Creatine kinase ( +23% at LOAEL) - 0.02 Serum lipoprotein levels (no quantification possible because data are expressed as ratio derived from NMR spectra, no statistics to determine LOAEL) PFDoDA 110-day RDT study Kidney: liver, nervous (95%) Rat (SD) Renal protein level of pyruvate carboxylase ( 10-18%) - Renal protein level of isovaleryl coenzyme A dehydrogenase, malate 0.05 system, immune system, n/sex/group = 6 (m) dehydrogenase 1 and dihydrolipoamide S-acetyltransferase 0.05 0.2 haematopoietic system, thyroid, Remarks endocrine system Exposure: oral (gavage), vehicle: 0.2% Study to assess MOA of nephrotoxicity: disorders in glucose and Tween-20) amino acid metabolism may contribute to nephrotoxicity; 2u globulin may play a role in protecting the kidneys from PFDoDA toxicity 66 (Ding et al., 2009) (Zhang et al., 2011) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 0.05, 0.2, 0.5 mg/kg bw/d NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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) - Exposure: oral (gavage), vehicle: 0.2% Tween-20) Doses: 0, 0.2, 0.5 mg/kg bw/d Remarks Study to assess MOA of hepatotoxicity: chronic PFDoDA exposure may inhibit insulin signal pathways and inhibition of GSK3 might contribute to the observed increases of lipid levels in the liver Kidney, nervous 0.2 system, immune 0.2 system, haematopoietic 0.2 system, thyroid, endocrine system (Zhang et al., 2013) PFDoDA (97%) PFDoDA 42-day RDT study, OECD TG 422, 14 day recovery (0, 2.5 mg/kg bw/d) Rat (SD) n/sex/group = 7 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) 110 day RDT study Body weight ( -33% at LOAEL) Liver: Rel. liver weight ( +15-20% at LOAEL) Hepatic hypertrophy Hepatic necrosis Inflammatory cholestasis Kidney: Rel. kidney weight , abs. (m only) Immune system: Abs. spleen weight ( -14-38% at LOAEL) White blood cells in recovery group (m only, -20%) Thyroid: Abs. thyroid weight (m, -55% at LOAEL) Haematopoietic system: Reticulocytes (m only, -63% at LOAEL) Mean corpuscular haemoglobin concentration (m only, +4%) Clinical chemistry: Serum ALP (m only, +54% at LOAEL) Serum total cholesterol (m only, -33% at LOAEL) Serum glucose (m, -27% at LOAEL) Globulin-2 (m, -14% at LOAEL) Other effects: Pancreatic zymogen granules (m only, 5/7 animals at HD/LOAEL, 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) Spermatid and spermatozoa counts Body weight ( -5.5% at LOEL) 0.5/0.5 0.1/0.1 0.5 0.5 0.5 0.5 0.5/0.1 - 0.5 0.5 0.5 0.1 - 0.5 0.1 0.5 0.5 0.5 0.2 2.5/2.5 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 Kidney, nervous (Kato et al., 2015) (Shi et al., 67 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 95% Rat (SD) Endocrine system: Serum testosterone ( -44% at LOEL) n/sex/group = 6 (m) Reproductive tissue: Cast-off cells in some seminiferous tubules in testes (no quant) Testicular gene expression of StAR ( -35% at LOEL) Exposure: oral (vehicle: Tween-20) Testicular gene expression of P450scc (only at 0.05) Testicular gene expression of IGF-I ( -25% at LOEL) Testicular gene expression of IGF-IR and IL-1 ( -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) NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) 0.05 0.2 0.2 0.5 - 0.02 0.02 0.05 0.05 0.2 0.2 0.5 Key parameters / targets not addressed system, immune system, haematopoietic system, thyroid Serum/tissue concentrate- Reference ion of PFAS /metabolites (time of sampling) 2009) PFDoDA 14-days RDT Body weight ( -25% at LOEL) 1 5 Kidney, nervous 95% Rat (SD) Clinical chemistry: Total serum cholesterol ( +38.5% at LOEL) system, immune 5 10 system, n/sex/group = 6 (m) Endocrine system: Serum LH ( -30% at LOEL)(FSH ns) haematopoietic 5 10 system, thyroid Testosterone ( -50% at LOEL) 1 5 Exposure: oral (gavage, vehicle: Estradiol (only at 5 mg/kg bw/d, -50%) Tween-20) Reproductive tissue: Apoptotic features present in Leydig cells, Sertoli cells and 5 10 spermatogenic cells Doses: 1, 5, 10 mg /kg bw/d Abs. testis weight ( -22% at LOEL) 5 10 Rel. testis weight ( +36% at LOEL) 1 5 Testicular gene expression of SR-B1 ( -60% at LOEL) 1 5 Testicular gene expression of StAR ( -50% at LOEL) 1 5 Testicular gene expression of P450scc ( -90% at LOEL) 1 5 (Shi et al., 2007) PFDoDA 110-day RDT study Liver: Kidney, nervous Rat (SD) Hepatic SOD activity TBARS in liver 0.2 0.5 0.2 0.5 system, immune system, n/sex/group = 4-10 (m) Hepatic GPX activity mRNA of PPAR/Cyp4A1 0.2 0.5 0.05 0.2 haematopoietic system, thyroid, mRNA of mitochondrial acyl-CoA-thioesterase 1 and hydroxyacyl- endocrine system Exposure: oral (gavage), vehicle: 0.2% CoA-dehydrogenase - 0.05 Tween-20) (Liu et al., 2016) 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 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks NO(A)EL LO(A)EL (mg/kg (mg/kg bw/d) bw/d) Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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 Rat (SD) n/sex/group = 12 (m+f) Exposure: oral (vehicle control,0.5% carboxymethylcellulose sodium in water) Dose groups: 0, 1, 3, 10 mg/kg bw/day Dosing regime All animals: 14 day pre mating Males: 42 day Females: during lactation up to 5 days after parturition (app. 42 days i.e. 14 premating, 1-2 days mating 21-23 gestation, plus 5 days) Recovery Group: high dose for 42 days without mating and vehicle control, followed by a 14 day recovery period 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) Body weight (f, -9% at LOAEL throughout entire lactation 3 period) Food consumption (f, day 5 and 10 gestation; day 4 lactation) 3 Liver: Abs. liver weight (m, +22% at LOAEL, also in recovery group) 1 Rel. liver weight (m/f, +19/+11% at LOAEL, also in recovery 1/3 groups) Centrilobular hepatocyte hypertrophy (m/f, also in recovery 1/3 groups) Microgranulomas (f, also in recovery group; m: microgranulomas - in treatment and control group) Focal necrosis in one female Kidney: Abs. + rel. kidney weight (m/f, ns) Immune system: Thymic cortex atrophy (m: NA; f: at LOAEL) 3 Abs.+rel. thymus weight (m/f, ns) Abs.+rel. spleen weight (m/f, ns) White blood cell count (m/f, ns) Thyroid: Follicular cell hypertrophy (m; f: NA) 1 Haematopoietic system: 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: -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 All major organs and tissues have 3 been included, as listed in the 10 manuscript 10 3 3/10 3/10 10 10 3 10 3 10/10 10 10 10 (HirataKoizumi et al., 2015) 69 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 (%), EC/CAS, formula study design, species, route of Observed effects exposure, doses (guideline/similar to guideline/non-guideline) Remarks Serum inorganic phosphorus (m, +8% only in recovery group; f: ns) 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) Hindlimb grip strength (m, -20-25%) Urinary parameters (ns) Reproductive/developmental toxicity: Body weights in male and female pups No effects on estrous cycle, copulation index, fertility index, gestation length, Corpora lutea, implantation sites, delivered pubs, sex ratio NO(A)EL (mg/kg bw/d) 1 3 3 LO(A)EL (mg/kg bw/d) 3 10 10 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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 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 Rat (SD) n/sex/group = 12 (m+f) Exposure: oral (vehicle control,0.5% carboxymethylcellulose sodium in water) Dose groups: 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 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) Liver: Abs. liver weights (m, +19% at LOAEL, also in recovery group; f: 20 ns) Rel. liver weights (m, +30% at LOAEL, also in recovery group; f: 20 ns) Centrilobular hepatocellular hypertrophy (m/f, m also in recovery 20 group) Centrilobular fatty changes 20 Kidney: Abs. + rel. kidney weight (m/f, ns) Immune system: Abs.+rel. thymus weight (m/f, ns) Abs.+rel. spleen weight (m/f, ns) White blood cell count (m/f, ns) Thyroid: Rel. thyroid weight (m, +26% at LOAEL, not in recovery group; f: 4 ns) Endocrine system: 100 100 100 100 100 100 20 (HirataKoizumi et al., 2015) 70 Commented [A22]: How important is it to fill in this? Most relevant for guideline studies deviating form guideline? Formatted: Spanish (Spain) Formatted: Spanish (Spain) 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) Remarks bw/d) recovery after treatment) T3 4, (m: ns; f, . --17% at LOAEL, not in recovery) .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 PFHpDA: Perfluoroheptadecanoic acid (Syn:, CAS no: EC no: , Mol. formula: C17HF33O2, MW: ) LO(A)EL (mg/kg bw/d) 4 100 100 100 100/20 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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 groups) Food consumption 4, at day 5 lactation Food consumption 4, at day 35 and 42 Liver Abs. liver weight I' (m/f, ,-.-. +45/+29% at LOAEL, also in recovery group) Rel. liver weight l' (m/f, .. +42/+35% at LOAEL, also in recovery group) Centrilobular hepatocellular hypertrophy (m/f, also in recovery group) Immune system Thymic atrophy of the cortex (2 animals/24) Abs. thymus weight 4, (m, --40% at LOAEL, not in recovery group; f: 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) 200 200 200 40/200 40 40/200 200 1000 1000 1000 kidney, nervous system, thyroid, endocrine system 200/1000 1000 (HirataKoizumi et al., 2012) 71 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks All animals: 14 day pre mating Haematopoietic system: Males: 42 day Females: until 5th day of lactation (4256 days) Erythrocytes, haemoglobin (m) Reticulocyte ratio m,m: recovery group Prothrombine time (f), Basophiles (f) Clinical chemistry: Albumin protein fraction (m, +15% at LOAEL, also in recovery group; f: only in recovery group) 1-globulin protein fraction (m/f, -41/-23% at LOAEL, remains reduced in recovery group) 2-globulin fraction (m+f, ns) -globulin protein fraction (m/f, -17/-10%, only in recovery group) -globulin protein fraction (m/f, +68/+59% at LOAEL, not in recovery group) AST (m+f, ns) ALT (m, +53% at LOAEL, f: ns) ALP (m/f, +88/+72% at LOAEL, remains increased in m in recovery group) Total bilirubin (m, +167% at LOAEL, but not in recovery group, f: ns) BUN (m/f, +41/+41% at LOAEL, not in recovery groups) Total cholesterol (f, -25%, not in recovery group; m: ns) Triglycerides (m, -67% only in recovery group; f: ns) Other effects pancreatic zymogen granules (m: tendency, f: significant) Rel. brain weight (m/f, +25/+13% at LOAEL, m also in recovery group) Reproductive and developmental findings: No significant differences in delivery, live birth and viability indices between control and PFODA treated groups, but a decreasing tendency in the HD-group. body weight of male and female pups NO(A)EL (mg/kg bw/d) 40 200 40 200 40 200/40 200 200 200 200 200 200 200 200 200 LO(A)EL (mg/kg bw/d) 200 1000 200 1000 200 1000/200 1000 1000 1000 1000 1000 1000 1000 1000 1000 Key parameters Serum/tissue concentrate- Reference / targets not ion of PFAS /metabolites addressed (time of sampling) 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 Commented [A22]: How important is it to fill in this? Most relevant for guideline studies deviating form guideline? 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Perfluoroalkyl carboxylic acids (PFCAs) Reference 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) PFBA, 17-days perfluorobuty GD1-GD17 rate, ammonium Mouse (CD1; timed-pregnant) (98%) Maternal n/group = 30 (3 blocks) Offspring sample size? Maternal maternal liver weight (30% est. Fig 2; reversible) 35 175 Fertility full litter resorptions (26.9%/350 mg/kg versus 6,8% in control) 175 350 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 350 mg/kg pregnant dams: 2.49 g/ml serum 0.96 g/g liver Pups PND1 0.37 g/ml serum 0.24 g/g liver (Das et al., 2008) Exposure: oral (gavage) Developmental pups liver weight PND1 (10-20% est. Fig 5; no longer observed at PND10) Doses: 0, 35, 175, 350 mg/kg slightly delayed eye opening (1-1.5 days) bw/d delayed vaginal opening (2-3 days) delayed preputial separation (350/0 mg/kg 33% vs 7%) 35 175 Pups PND10 0 35 1.12 g/ml serum 35 175 0.04 g/g liver 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) PFHxA ammonium 93.4% purity 12-day GD 6-18 (for both experiments?) Mouse (CD1; timed-pregnant) Maternal: n/group = 20 (for both experiments?) Exposure: oral (gavage) Two phases i.e. two experiments: Experiment (phase) 1 Doses: 0, 100, 350, 500 mg/kg bw/d Maternal maternal mortality (0: 2/20; 100: 6/20; 350: 1/20; 500: 3/20) Fertility Postnatal mortality (PND 0) (0:0/217; 100: 0/250; 350: 3/232; 500:21/150) Postnatal mortality (PND 1-4) (0: 2/217; 100: 2/250; 350: 25/229; 500; 20/129) decreased litter size neonatal mortality Developmental weight gain during lactation relative liver weight in F1 350 500 100 350 350 500 350 500 (Iwai and Hoberman, 2014) Experiment (phase) 2 Phase 2 increased postnatal mortality (PND 0) (0: 0/249; 7:0/211; 35: 0/232; 175: 4/238) 73 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 7, 35, 175 mg/kg bw/d maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Reference Statistical re-evaluation of Iwai and Hoberman 2014 confirmed 175 mg/kg as NOAEL for the reported pup mortality PFHxA (Na-PFHxA) comprehensive toxicological evaluation including A) One generation reproduction study similar to OECD TG 415 P0 70d prior to mating until weaning 126d (f), 110d (m) Rat (SD) Maternal reduced maternal body weight gain (10-20%) Developmental reduced pup body weight (10-20%) 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) Maternal reduced maternal body weight gain (19%) Developmental decreased fetal weight (10%) n/sex/group = 20 Exposure: oral (gavage) no detectable effects related to reproduction 74 175 350 100 500 100 500 100 500 100 500 (Iwai et al., 2019) (Loveless et al., 2009) 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Doses: 0, 20, 100, 500 mg/kg bw/d Reference 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 ammonium 98% purity GD1 - birth Mouse (CD1; timed-pregnant) (no information on number per dose) Exposure: oral (gavage) Doses: 0, 1, 3, 5, 10, 20,40 mg/kg Maternal liver weight (2-fold < 3 mg/kg) 0 decreased maternal weight gain 5 Developmental neonatal mortality (Very high in the 20 mg/kg (60%) and 40 mg/kg (90%) dose groups) growth retardation and and decreased weight among surviving pups enlarged frontanel, reduced ossification (sternebrae, calvaria), minor tail and limb defects 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 P0: (only A) increased neonatal mortality P0: compromised weaning induced mammary involution PND22 (A) 0 P0: compromised weaning induced mammary involution PND22 (B) 0 B: pregnant mice (CD-1) F1: reduced developmental mammary scores (A) during gestation (GD1-17/birth) F1: reduced developmental mammary scores (B) 0 mg/kg (n=7) 1 mg/kg (n=10) Exposure: oral (gavage) 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 75 1 10 3 3 5 1 1 0.00045 0 0 0 0 1 1 For determination of serum (Lau et al., levels: 2006) rats 10 mg/kg 20 d 5 mice 20 mg/kg 7 or 17d mice 20 mg/kg 17 d 199 g/ml (m) 5 171 g/ml (f) 10 rats 10 mg/kg 20d 111 g/ml (m) 0,69 g/ml (f) Group only exposed to 5ppb (White et 5 PFOA (B1) al., 2011) 1 0.00045 p0->F2 P0 weaning: 74,8 ng/ml F1 PND22 21.3 ng/m F1 PND63 66.33 ng/m F1 dams weaning 86.9 ng/ml F2 pups PND22 26.6 ng/ml F2 pups PND63 68,5 ng/ml 1 0.00045 5 5 Group P0 1 mg/ml/ 5ppb P0>F2 (B2) P0 weaning: 4772.0 ng/ml 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 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PND 22 (ns) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Reference PFOA 90% purity PFOA PFOA 17-days, GD1-17 Mouse (ICR; timed-pregnant) Maternal sample size: n/group = 15-19 Exposure: oral (gavage) Doses: 0, 1, 5, 10 mg/kg bw/d Two generation study EPA OPPTS 8703800 P0 70 day prior to mating (m) or until weaning (f) Rat (SD) 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 timed pregnant mice (CD-1) Litter sample size: n/group = 11-13 Exposure: oral (gavage) 0, 1, or 5 mg/kg bw/d sacrifice timepoint GD11.5 or GD17.5 Maternal liver weight (relative weight 5.4-11.4%) Increased GGT, AST, ALP Reduced globulins, triglycerides and cholesterol Developmental reduced birth weight neonatal mortality (100%, 10 mg/kg) Parental no reproductive endpoints affected in any generation Developmental (F1) birth weights in relationship with reduced viability Reduced viability not observed in F2 Preputial separation and vaginal opening somewhat delayed Maternal abs liver weight (E11.5) (0: 2.2g; 1: 2.9g, 5: 4.5g) rel liver weight (E11.5) (0:5.9%; 1:7.4%, 5: 11.0%) enlarged hepatocytes (E17.5) abnormal ultrastructure of liver cells (increased peroxisomes/mitochondria) 76 1 5 5 10 5 10 1 1 10 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 (Yahia et al., 2010) 5 5 (Butenhoff et al., 2004) 30 0 1 0 1 0 1 0 1 (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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks dosing GD1.5 until 11.5 or GD 1.5 until Triglycerides (E 11.5) (1 mg/kg 37%, 5 mg/kg 58%) 17.5 Glu (5 mg/kg E17.5: 20% lower) AST (5 mg/kg E17.5: up 100%) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 0 1 1 5 1 5 Fertility embryo weight 1 5 placental lesions (nodules, labyrinth congestion or atrophy 0 1 PFOA GD1-17 Maternal Ammonium Mouse (PPAR KO -/- or 129S1/SvlmJ Maternal liver weight (WT) (more than 2-fold at 5 mg/kg) 1 5 salt (APFO) WT), timed-pregnant Maternal liver weight (PPAR-KO) (more than 2-fold at 5 mg/kg) 1 3 Maternal liver weight not dependent on PPAR 98% purity Number of litters highly variable n/group = 4-22 litters for individual Fertility doses litter loss wt (100%) (1 mg/kg: 70%) 1 5 litter loss wt PPAR KO -/- (100%) (5 mg/kg: 86%) 10 20 Exposure: oral (gavage) Comparable embryo lethality in WT and PPAR KO mice at 5 mg/kg Doses: 0.1, 1, 3, 5, 10 or 20 mg/kg (100% versus 86%) bw/d Embryo lethality:PPAR-independent 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 (PPAR-KO) (0 mg/kg: 92% vs. 1 mg/kg > 90%, 3 mg/kg: 87%) neonatal mortality (WT: Fig 2A in publication) neonatal mortality (PPAR-KO: Fig 2B in publication) neonatal mortality PPAR-dependent delayed eye opening (WT) ( 1 day) delayed eye opening (PPAR-KO) 0.3 3 0.6 - 0.3 0.6 1 3 Reference (Abbott et al., 2007) 77 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFOA GD1 - GD17 Developmental Male offspring was analyzed at PD 21 neonatal mortality (PD7) and PD70 decreased number of Leydig cells Mouse (Kunming) altered ultrastructure, markedly increased intracellular substance maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 2.5 5 1 2.5 1 2.5 n/sex/group = 10 (litters or male pups?) Exposure: oral (gavage) Expression of imprinted Dlk-Dio3 gene cluster no difference in testicular index 1 2.5 PFOA ammonium salt (APFO) >98% purity Doses: 0, 1, 2.5 or 5 mg/kg bw/d 7-17 days, GD1-GD17 (full gestation exposure) or GD10-GD17 (late gestation exposure) Mouse (CD-1), timed-pregnant Maternal n/group = 13 (both experiments) Litters n/group = 13 (equalized to 5 m and 5 f 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) Developmental no effects on offspring body weight in both experiments (f+m) abs/rel liver weight of offspring in full/late gestation exp. (f+m) delayed epithelial growth in the mammary gland (full-gestation exp: PND 63 and 84, on PND 63 no control comparison, but comparison to historical controls...) delayed epithelial growth in the mammary gland (late gestation exp: PND1-PND14/PND 14) overall developmental scores in both experiments (full/late gestation exp) 0.3 0.3 0.1 0.3/ 0.01 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 Reference (Song et al., 2018) (Macon et al., 2011) 78 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFOA 17 days, Developmental ammonium GD1-GD17 net body weight (body weight-liver weight) (only CD-1) salt (APFO) Mouse (CD-1 and C57Bl/6); timed- no effect on abs liver weight (both strains) >98% purity pregnant rel liver weight at PND 21 (but not at PND35 and 56)(only CD-1 mice) Maternal sample size: estradiol and progesterone (ns in both strains) 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) Litter sample size mammary gland developmental scores reduced on PND 35 and 56 (equalized to 10 pups per dam, each 6- (tbc) (CD-1) 7 f + 3-4 m) mammary gland developmental scores reduced on PND 21 and 61 (tbc) (C57Bl/6) Exposure: oral (gavage, in water) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 1 1 0.01 0.3 Reference (Tucker et al., 2015) 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 97% purity 90-day study, 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 0.2 0.5 LOEL) serum cholesterol ( -33% at LOEL) 0.2 0.5 serum testosterone ( -31% at LOEL) 0.2 0.5 Litter size ( -42% at LOEL) (reproduction assay) 0.2 0.5 testicular proteins/mRNA involved in steroidogenic processes 0.2 0.5 (StAR/Cyp11A1/3-HSD/17-HSD) ( -47%/-49%/-35%/-64% at LOEL) Testes lipid peroxidation ( +41% at LOEL) 0.2 0.5 Antioxidative enzymes (SOD, CAT) ( -22%/-27% at LOEL) - 0.2 number of proliferating testicular cells (PCNA) ( -28% at LOEL) 0.2 0.5 Testes apoptosis (Caspase-3 staining) ( +35% at LOEL) 0.2 0.5 (Singh and Singh, 2019b) 79 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFNA 14-day study, Males 97% purity PND25 -PND38 (prepuberty) Mouse (Parkes strain) Body weight gain Fertility intratesticular/serum testosterone ( -20%/-72% at LOEL) Rel no. affected seminiferous tubules ( +424% at LOEL) n/sex/group = 10 (m) testicular proteins/mRNA involved in steroidogenic processes (SF- 1/StAR/Cyp11A1/3-HSD/17-HSD) ( -23%/-57%/-50%/-45% /- Exposure: oral (gavage) 46% at LOEL) Testes lipid peroxidation ( +74% at LOEL) Antioxidative enzymes (SOD/CAT/GST) ( -31%/-35%/-34% at Doses: 0, 2, 5 mg/kg bw/day LOEL) number of proliferating testicular cells (PCNA) ( -13% at LOEL) Testes apoptosis (Caspase-3 staining) ( +100% at LOEL) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 2 5 - 2 - 2 - 2 - 2 - 2 - 2 - 2 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 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/Cyp11A1/3-HSD/17-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/ 5/2/5/ 5/ 2 5 -/ 2 2/ 5 2 5 PFNA 97% purity Doses: 0, 2, 5 mg/kg bw/day 14-day study, PND25 -PND38 (prepuberty) Mouse (Parkes strain) n/sex/group = 10 (m) Exposure: oral (gavage) Males Abs/rel liver weight ( +100/42% at LOEL) - 2 Hepatocellular hypertrophy - 2 PPAR expression (80-fold change at LD, only 5-fold change at HD) - 2 Hepatic liver peroxidation ( +26% at LOEL) - 2 Fertility Testicular glucose ( -22% at LOEL) - 2 Testicular lactate ( -1% at LOEL) - 2 Serum cholesterol ( -19% at LOEL) 2 5 Fasting blood glucose ( +16% at LOEL) - 2 80 Reference (Singh and Singh, 2019d) (Singh and Singh, 2019c) (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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Doses: 0, 2, 5 mg/kg bw/day maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Reference PFNA (97%) 14-day-study Fertility Rat (SD) Serum testosterone ( -85% at LOEL) Serum estradiol ( +104% at LOEL) 3 5 3 5 n/sex/group = 6 (m) Apoptotic cells in testes ( +367% at LOEL) Fas gene ex ( +90% at LOEL) 1 3 3 5 Bax gene ex ( +36% at LOEL) 3 5 Exposure: oral (gavage) Bcl-2 gene ex ( -27% at LOEL) Vacuolization between Sertoli cells and spermatogonia 1 3 1 3 Testicular WT1 protein levels ( +59% at LOEL) - 1 Doses: 0, 1, 3, 5 mg/kg bw/d Testicular Transferrin protein levels ( -33% at LOEL) - 1 Serum MIS ( +22% at LOEL) 3 5 Serum inhibin B ( -10% at LOEL) - 1 PFNA GD 1-20 Maternal Purity and salt not specified Rat (SD) Maternal (pregnant females): Body weight b( -25%/-11% at GD10/GD21) Fertility Birth weight ( -10% for females) Developmental n/sex/group = not specified, but since Systolic blood pressure at 10 weeks of age ( +7/10% for m/f) there were 18-19 litters, there were Systolic blood pressure at 26/56 weeks of age (ns) probably 18-19 dams... (?) Nephrons per kidney (m) ( -19%) Offspring: n/group = 18-19 litters for birth weight n/group = 10-12 pups/litter (litter size was standardized) n/group/sex = 1-2 per litter (not specified how many litters) for systolic blood pressure n/group = 5 (1 per litter from 5 litters) for nephrons (males only) Remark: Offsping was weaned by non-treated foster-dams! No maternal toxicity effects and no lactational effects Also arsenic trioxide and nicotine bitartrate were also tested as comparative substances (to PFNA. A second experiment with separate a control comparing Dex (as positive control; known to increase blood pressure), Atrazine, and PFOS, is also published in this paper. Body weight gain in PFNA treated animals below those treated with arsenic. Lowest bw gains were observed with nicotine. Exposure: oral (gavage) (only dams) 81 (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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites Doses: 0, 5 mg/ kg bw/d Reference PFNA >98% purity 28-day RDT study, NTP study, similar to Fertility OECD TG 407 Plasma testosterone in m ( -81% at LOEL) Rat (SD) Plasma testosterone in f ( +30% at LOEL) L. Cauda Epididymis weight (m, -11% at LOAEL) n/sex/group: 10 L. Epididymis weight (m, -7% at LOAEL) Testis weight (m, -7% at LOAEL) Sperm count (106/g cauda epididymis) (m, -18% at LOAEL) Exposure: oral (gavage, in distilled water) Germinal epithelium degeneration Interstitial cell atrophy in testes Seminiforous tubule spermatid retention 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 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 PFNA 17 days ReproDevTox study (GD1-17), Maternal 97% purity non-guideline study Mouse (CD-1) Maternal body weight at GD13 ( -30% at 10mg/kg bw/d) 5 Maternal body weight between GD11-GD17 ( +10% at LOEL) 1 Abs/rel liver weight (non-pregnant) ( +83%/88% at LOEL) - Maternal sample size: Abs/rel liver weight (pregnant) ( +45%/41% at LOEL) - Abs/rel liver weight (post-weaning) ( +33%/19% at LOEL) - n/group = 1-10 (for pregnancy outcome) n/group = 19-27 (maternal body weight gain) n/group = 5-15 (liver weights) Offspring sample size: Fertility Pregnancy not carried to term (full litter resorption) at LOEL 5 Pregnancy outcome (Implants per litter, abs/rel live foetuses per litter, fetal weight, skeletal or visceral examinations) (ns) Developmental 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) weight and neonatal survival) Postnatal body weight at PND 7/14/21 ( -17/24/23% at LOEL; n/group=6-13 litter (offsping body and effects persist in male pups until PND 287, but recovers in female liver weight & developmental delay) pups after PND 49) Rel postnatal liver weight at PND10/24 ( +37/27% at LOEL) 82 10 3 1 1 1 10 - 3 1 - (NTP, 2019b) Serum concentrations (g/ml): (Das et al., Pregnant at term: 2015) 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 1 Post-weaning: 5 3 Control: 0.020 0.001 1 mg/kg bw/d: 10 3 mg/kg bw/d: 15 1 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Rel postnatal liver weight at PND 42 ( +18% at LOEL) Exposure: oral (gavage, vehicle: deioinized water) Delayed development (Eye opening +13%, preputial separation +11%, vaginal opening +10% at LOEL) Gene expression heatmap authors: "PFNA induced a clear PPAR- Doses: 0, 1, 3, 5, (10, only till GD13) mg/kg bw/d dependent gene expression signature in both fetal and neonatal mouse liver." (Rather complex heatmap. PPAR not measured directly, but only PPAR dependent genes. maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 1 3 1 3 Liver concentrations (g/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 Remarks: Since overt toxicity was Remarks: Increased liver weight of offspring persistent into adulthood. indicated at 10 mg/kg bw/d, this group Absolute liver weights were significantly increased in the 1, 3 and 5 was sacrificed at GD13 and not mg/kg dose groups. included for further analysis in this However, this effect was not dose dependent, as the 5 mg/kg group study. was less affected 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." 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 (g/g): Control: 0.011 0.007 1 mg/kg bw/d: 10 3 mg/kg bw/d: 35 5 mg/kg bw/d: 70 Reference Serum offspring (g/ml) (at PND1PND10PND24): 1 mg/kg bw/d: 20101 3 mg/kg bw/d: 50205 5 mg/kg bw/d: 758025 Liver offspring (g/g) (at PND1PND10PND24PND42 PND70): 1 mg/kg bw/d: 556020105 3 mg/kg bw/d: 150150553020 83 Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) Formatted: Norwegian (Bokml) 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 5 mg/kg bw/d: 2052101753540 Reference Formatted: Norwegian (Bokml) PFNA (97%) GD 1-PND21 (42-days for nonpregnant Maternal females) Body weight (ns) Mouse (129S1/SvlmJ WT and PPAR Abs/rel liver weight (non-pregnant WT) ( +73/72% at LOEL) KO on 129S1/SvlmJ background (Ppara- Abs/rel liver weight (pregnant WT) ( +26/12% at LOEL) tm1Gonz/J)) Abs/rel liver weight (non-pregnant KO) ( +46/26% at LOEL) Fertility - 0.83 0.83/- 1.1/0.83 1.1/0.83 1.5/1.1 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 Exposure: oral (gavage) Pregnancy rate (KO) 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) 84 Serum PFNA (g/ml) in females with no live pups (WT/KO): (Wolf et al., 2010) 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/ 1.1 83.4 2.93 2 2 Serum PFNA (g/ml) in 2 females with live pups 0.83 (WT/KO): Control: 0.022 0.004/ 2 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 (g/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 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 (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal NO(A)ELLO(A)EL offspring NO(A)EL LO(A)EL Serum/tissue concentration of PFAS /metabolites 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 Reference 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 tox) Parental (F0+F1) component Rat (SD) PFNA) (15.2% Body weight (F0, m) (study weeks 0-7) (ns) Body weight (F0, m) (study week 7-18) ( -25% after study week 18 in water, doses already n/sex/group = 30 (F0 and F1 corrected) generation) at LOEL) Food consumption (m, pre-mating weeks 0-10) (data not shown) Abs/rel liver weight (F0, m) ( +22/22% at LOEL) CAS No. of mixture: 72968-38-8 Exposure: Oral (gavage) Abs/rel liver weight (F0, f) ( +16/14% at LOEL) Abs/rel liver weight (F1, m) ( +16/17% at LOEL) Abs/rel liver weight (F1, f) ( +16/14% at LOEL) 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) 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 0.6 0.6 0.125 0.6 0.125 0.6 0.125 0.125 0.125 0.025/0. 125 0.6 0.025/0. 125 0.025 0.125/0. 6 0.6 0.6 0.6 0.6 Serum concentrations Stump et analysed for C8 (PFOA9, C9 al. 2008 (PFNA), C11 (PFUnDA) and C13 (PFTrDA) (ng/ml): Cmax (peak 0-16 h) F0 females (Day 64): 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 (?) 0.125 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks Body weight (F1, m) (post-weaning week 1-10) (ns) Body weight (F1, m) (post-weaning week 11-22) (-32% in post- 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) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 0.6 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. Reference 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, 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 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 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 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. EFSA 2020 (summary at the end of Repro 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFDA 4/10-day DevTox Study, non-guideline Maternal (GD 10-13): 96% Mouse (C57BL/6N) maternal body weight gain ( -61% at LOEL) Abs/rel liver weight ( +10/3% at LOEL) n/group (maternal) = 10-13/10-14 Maternal (GD 6-15): Maternal body weight gain ( -92% at LOEL) n/group (litters) = 10-13/7-14 Abs/rel liver weight ( +61%/+127% at LOEL) maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 8 16 0.5/0.25 1/0.5 3 6.4 0.3 1 n/group (foetuses) = 83-106/23-53 Fertility (GD 10-13): Live foetuses per litter ( -32% at LOEL, but ns) 16 32 Exposure: oral (gavage, in corn oil) Resorptions per litter (ns) ( +170% at LOEL) 16 32 Fertility (GD 6-15): Doses: 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 Live foetuses per litter ( -46% at LOEL) mg/kg bw/day on gestation days (GD) Resorptions per litter ( +344% at LOEL, but p=0.06) 6.4 12.8 6.4 12.8 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) Developmental (GD 10-13): Fetal body weight ( -10% at LOEL) Developmental (GD 6-15): Fetal body weight ( -3% at LOEL) 0.25 0.5 0.03 0.1 Absence of fifth sternabrae (15% of examined foetuses at LOEL) 3.0 6.4 Delay in braincase ossification (overall) (26% of examined foetuses at - 0.03 LOEL) Delay in phalanges ossification (18% of examined foetuses at LOEL) 3.0 6.4 PFDA (>98%) 28-day RDT study, NTP study, similar to Fertility OECD TG 407 Plasma testosterone in m ( -64% at LOEL) 0.625 1.25 Rat (SD) Plasma testosterone in f ( +32-355% at LOEL) L. Cauda Epididymis weight (m, -11% at LOAEL) 0.156 0.625 0.312 1.25 n/sex/group = 10 L. Epididymis weight (m, -10% at LOAEL) Testis weight (m, -11% at LOAEL) 0.625 1.25 1.25 2.5 Sperm number?(106/g cauda epididymis) (m, -30% at LOAEL) 1.25 2.5 Exposure: oral (gavage, in distilled Interstitial cell atrophy in testes (m, 80% incidence at LOAEL) 0.625 1.25 water) Reference (Harris and Birnbaum, 1989) (NTP, 2019b) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 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) Body weight (female satellite group) (day 38-41+ 14-day recovery; similar in males, but ns) abs. liver weight rel. liver weight Hepatocyte hypertrophy ( 58-52% incidence at LOAEL) rel./abs. spleen weight (m only, -19-23% at LOAEL) serum ALP, ALT (m only, +26% for ALT and +139% for ALP) serum urea nitrogen ( +46-61% at LOAEL) serum albumin (m only, -7% at LOAEL) total protein ( -10-11% at LOAEL) 0.3 - 0.3/0.3 0.1/0.3 0.3/0.3 0.3/0.3 0.3 0.3/0.3 0.3 0.3/0.3 Fertility (m/f) No effects on epididymis, testis/uterus, estrous cycle or delivery/nursing (ns) 1 1 1/1 0.3/1 1/1 1/1 1 1/1 1 1/1 Developmental No abnormal findings in general appearance of pups Body weights (PND0) (m/f) ( -13/12% at LOEL) 0.3 1 Body weights (PND4) (m/f) ( -19/16% at LOEL) 0.3 1 Reference (Takahashi et al., 2014) 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) Fertility abs. L. Epididymis weight (m, -32% at LOAEL) spermatid and spermatozoa counts (m) discontinuous dioestrus (f) 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 (Kato et al., 2015) 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) 0.2 0.5 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks 95% Rat (SD) Fertility Abs/rel weight of testis, prostate, seminal vesicle and vas deferens n/sex/group = 6 (m) (ns) Serum testosterone ( -44% at LOEL) 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-1 ( -25%/-30% at 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) PFDoDA 28 day RDT study Maternal 95% PND24 till PND52-55 (i.e 28-31-days, Body weight (-6% at LOEL) sample collection in dietrous stage, Fertility pubertal females) Serum total cholesterol (+32% at LOEL) Serum estradiol (-37% at LOEL) Rat (SD) Serum FSH+LH (ns) Ovarian gene expression (-35% for LHR, -40% for StAR, -36% n/sex/group = 8 (f) for P450SCC, -36% for ER- at LOEL) Ovarian gene expression ER- (-36% at LOEL) Exposure: oral (vehicle: Tween-20) Ovarian gene expression 17-HSD (+110% at LOEL) Remark: "P450SCC is a rate limiting enzyme responsible for the conversion of cholesterol to pregnenolone in estrogen biosynthesis." maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 0.05 0.2 0.2 0.5 - 0.02 0.02 0.05 0.05 0.2 0.2 0.5 1.5 3 1.5 3 1.5 3 1.5 3 0.5 1.5 - 0.5 Doses: 0, 0.5, 1.5, 3 mg/kg bw/d PFDoDA 95% 14-days RDT study Rat (SD) n/sex/group = 6 (m) Exposure: oral (gavage, vehicle: 20) Doses: 1, 5, 10 mg /kg bw/d Tween- Males Body weight ( -25.2% at LOEL) Fertility Total serum cholesterol ( +38.5% at LOEL) Serum LH ( -30% at LOEL)(FSH no effect) Testosterone ( -50% at LOEL) (Estradiol (only at MD, -50%)) Apoptotic features present in Leydig cells, Sertoli cells and spermatogenic cells Abs testis weight ( -22.4% 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) 1 5 5 10 5 10 1 5 5 10 5 10 1 5 1 5 1 5 1 5 89 Reference 2009) (Shi et al., 2009) (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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks PFDoDA 14-day RDT study Males Rat (SD) Body weight ( -21% at LOEL) Fertility n/sex/group = 8 (m) Abs. testis weight ( -8% at LOEL) Serum testosterone ( -82% at LOEL) Serum LH ( -36% at LOEL) Exposure: oral (gavage) Serum FSH ( -9% at LOEL) Gene expression of Leydig cell genes from testis ( -29% for Lhcgr, -47% for Cyp11a1 at LOEL) Doses: 0, 5, 10mg/kg bw/d Gene expression of Leydig cell genes from testis ( -57% for 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) PGC-1 signalling ( -35% at LOEL) maternal NO(A)ELLO(A)EL 5 10 5 10 - 5 - 5 - 5 - 5 5 10 - 5 5 10 offspring NO(A)EL LO(A)EL Serum/tissue concentration of PFAS /metabolites 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. Reference (Chen et al., 2019) 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 Parental Perfluorotetra reproductive/developmental toxicity Males: body weights on day 7 and day 14 (treated animals versus 3 10 decanoic acid (OECD TG 422) recovery group) Rat (SD) Females: body weight on day 4 of lactation Females: body weight during lactation period 1 3 3 10 n/sex/group = 12 Males: absolute and relative liver weight Females: relative liver weight 1 3 3 10 (HirataKoizumi et al., 2015) Exposure: oral (gavage, vehicle 0.5% carboxymethylcellulose sodium in water) Doses: 0, 1, 3, 10 mg/kg bw/day) Dosing regime Fertility No effects on estrous cycle, copulation index, fertility index, gestation length, Corpora lutea, implantation sites, delivered pubs, sex ratio Developmental effects Body weight of pups (m/f) (-14/13% on PND 1, -18/17% on PND4 at LOEL) 90 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites All animals: 14 day pre-mating Reference 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) PFHxDA, Repeated dose and Parental Perfluorohexa reproductive/developmental toxicity Males: body weights on day 35 and day 42 of administration 20 100 decanoic acid (OECD TG 422) period Rat (SD) Males: food consumption decreased at day 14 of recovery period 20 100 Females: food consumption GD 5 - 10 gestation; LD 4 20 100 n/sex/group = 12 Exposure: oral (gavage, vehicle 0.5% carboxymethylcellulose sodium in water) 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 (HirataKoizumi et al., 2015) 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 Observed effects (%), EC/CAS, exposure, doses (guideline/similar formula to guideline/non-guideline) Remarks maternal offspring Serum/tissue NO(A)ELLO(A)EL NO(A)EL LO(A)EL concentration of PFAS /metabolites 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) 200 Exposure: oral (gavage, vehicle 0.5% number of implantations (-15% at LOEL) 200 carboxymethylcellulose sodium in Total number of pups born (-28% at LOEL) 200 water) No of live pups on PND0/PND4 (-35/5% at LOEL) 200 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. 200 Dosing regime: All animals: 14 day pre mating Males: 42 day Developmental Body weight pups (m/f) (=-23/23% on PND0, -27/26% on PND1, 200 --28/26% on PND 4 at LOEL) Females: until 5th day of lactation (42- Body weight gain (m/f) (-37/32% at LOEL) 200 56 days) 1000 1000 1000 1000 1000 1000 1000 Reference (HirataKoizumi et al., 2012) 92 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 indication for neoplasia/hyperplasia 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 NO(A)EL LO(A)EL Reference (Klaunig et al., 2015) PFOA 24-months RDT study Rat (SD), n/sex/group = 50-65 Exposure: oral (dietary, ad libitum) Doses: 0, 30, 300 ppm; corresponding to approximately 0, 1.5, 15 mg/kg bw/d (0, 1.3 or 14.2 mg/kg bw/d for males and 0, 1.6 or 16.1 mg/kg bw/d for females) Neoplasia/hyperplasia Liver Liver adenoma: 6%, 2%, and 10% in males and 0%, 0%, and 2% in females in C, LD, HD Reproductive tissue Leydig cell adenoma (0, 4, 14% in C, LD, HD) mammary gland adenoma in females: 15%, 31%, and 11% in C, LD, HD fibroadenomas in mammary glands of females: 22%, 42%, and 48% in C, LD, HD Pancreas pancreatic acinar cell hyperplasia: 0%, 4%, 4% in C, LD, HD from original study; and after re-evaluation of male 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, 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." tissue 1.5/- - 1.5 1.5/- PFOA (98- 24-months RDT study 100%) Rat (Crl:CD BR (CD)) n/sex/group = 156 (males only) n/group (neoplasia/hyperplasia) = 76-80 Exposure: oral (dietary) Doses: 0, 300 ppm (equivalent to 13.6 mg/kg bw/d) 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)) - Pancreas Acinar cell hyperplasia (+290% (39% vs. 10% in C)) - Acinar cell adenoma (+800% (9% vs. 1% in C)) - Reproductive tissue Leydig cell hyperplasia (+39% (46% vs. 33% in C)) - Leydig cell adenoma (+267% (11% vs. 3% in C)) - 93 (3M, 1983) (Sibinski, 1987) 15/- - (Butenhoff 15 et al., 2012b) 15/- (Hardisty et al., 2010) (Caverly Rae et al., 2014) (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 PFOA (>98%) 107-weeks RDT study with perinatal and post-weaning exposure Rat (SD) n/sex/group: study 1: F0 females: 103 (C) or 36 (150 and 300 ppm groups); F1 rats: 60 m and 60 f; study 2: F0 females: 147, F1 males: 60 (no females) Exposure: oral (diet) Doses: study1: perinatal: 0, 150, 300 ppm; postweaning: m: 0, 150, 300 ppm ( 0, 16, 32 mg/kg bw/d), f: 0, 300, 1000 ppm ( 0, 30, 100 mg/kg bw/d) study 2: perinatal: 0, 300 ppm; post weaning: 0, 20, 40, 80 ppm ( 0, 1.1, 2.2, 4.6 mg/kg bw/d) Neoplasia/hyperplasia Non-neoplastic lesions were only observed in the liver and pancreas of male rats 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, 0/80: 0%, 0%, 14%, 22%; 300/0, 300/20, 300/40, 300/80: 0%, 2%, 10%, 24%) 1.1 higher incidences of hepatocellular carcinomas in the 300/80 ppm group compared to the 0/80 group Pancreas 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%) - occurrence of acinar cell adenomas and adenocarcinomas in the 0/1,000 and 300/1,000 ppm female groups (not statistically significant) 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 (predominately acinar cell adenomas) of the pancreas; there was no increase in the incidence of Leydig cell neoplasms observed in this study, possibly due to lower doses compared to two previous studies (3M, 1983; Biegel et al., 2001) PFOA (purest available analytical grade) 52-week RDT study to assess tumorpromoting activity of PFOA Rat (Wistar, 180 g) n/sex/group = 15 (m) Exposure: oral (diet, ad libitum) Neoplasia/hyperplasia 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; 0.015%: 4/12) 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 LO(A)EL Reference (NTP, 2019a) 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; triphasic protocol - 200 mg/kg diethylnitrosamine + 0.03% 2-AAF in diet for 2 weeks + single oral dose of CCl4 (2 mL/kg) positive control: phenobarbital (0.05%) PFOA increased the incidence of malignant hepatocellular carcinoma and also selectively induced the peroxisomal acyl-CoA oxidase activity and catalase activity 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 (%) PFDA or 8:2FTOH study design 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. 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. Observed effects 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. NO(A)EL PFOA, PFOS EFSA Opinion 2018: Risk to human health related to the presence of PFOS and PFOA in food PFOS PFOS was found to cause tumours in the liver of rats. Mechanistic studies suggested that the compound may act as a tumour promoter. 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. LO(A)EL Reference (EFSA, 2020b) 95