Annex A Tables 14 to 24
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Skip the menu of subheadings on this page.This is a paper for discussion. This does not represent the views of the Committee and should not be cited.
Table 14 In vitro toxicity studies for PFCAs – PFNA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFNA CAS No. 375-95-1. 97%. Long et al. (2013). |
GH3 cells. 3 x 103 cells/well (96 well plate). 3 expts. in quadruplicate. Non-GL study. GLP not stated. |
1 - 1 x 105 6 days. |
0.02% DMSO. 0.5 nM T3. LDH. Cytotoxic at >1 x 106 nM. |
Thyroid hormone-dependent cell growth (T-screen assay). |
Without T3 (agonistic response). ↓cell proliferation. LOEC = 1 nM. RPE at MOEC = -2.87% relative to T3. With T3 (competitive response). ↓ T3-induced cell proliferation. LOEC = 10 nM. RPE at MOEC = -1.89% relative to T3. |
PFNA has the potential to inhibit the function of the TH system. |
K2 Aim of the study was to assess in vitro the potential impacts of seven PFAS on TH activity using the rat pituitary tumour cell line GH3 expressing intracellular TR and responding to physiological concentration of TH by proliferation (named as T-screen). T-screen assay exposure duration 6 days. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFNA CAS No. 375-95-1 97%. Long et al. (2013). |
Hepa1.12cR cells. 6 × 104 cells/per well 2 expts. in triplicate. Non-GL study. GLP not stated. |
1 - 1 x 105 Duration not given. |
0.02% DMSO. 60 pM TCDD. LDH. Cytotoxic at >1 x 106. |
Activation / inhibition of AhR. (AhR transcriptional activation bioassay). |
Without TCDD (agonistic response). No observed effect. With TCDD (antagonistic response). No observed effect. |
No effect on AhR. |
K1 No data on the number of cells plated. Aim of the study was to assess in vitro the potential impacts of seven PFAS on AhR function. The AhR transcriptional activity was determined by AhR-luciferase reporter gene bioassay using the stably transfected mouse Hepa1.1 2cR cell. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFNA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 1977 ± 1698 nM. RP = 0.016 relative to T4. TTRmutK15G. IC50 = 6.00 x 103 nM.
|
Compared to WT TTRT4 exhibited much weaker binding affinity to TTRmutK15G. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFNA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G 1000 nM. 3 expts in triplicate. Non-GL study. GLP not stated. |
Human WT TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. IC50 = 2.26 x 105 nM. TBGmutR381G. IC50 = 7.00 x 104 nM.
|
Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFNA CAS No. 375-95-1. 97%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104 . Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 2.74 x 103 nM. RP = 0.022 relative to T4. |
Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 15 In vitro toxicity studies for PFCAs – PFDA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFDA CAS No. 375-76-2. 98%. Long et al. (2013). |
GH3 cells. 3 x 103 cells/well (96 well plate). 3 expts. in quadruplicate. |
1 - 1 x 105 6 days. |
0.02% DMSO. 0.5 nM T3. LDH. Cytotoxic at >1 x 106 nM. |
Thyroid hormone-dependent cell growth (T-screen assay). |
Without T3 (agonistic response). ↓ in cell proliferation. LOEC = 10 nM. RPE at MOEC = -3.13% relative to T3. With T3 (competitive response). ↓ T3-induced cell proliferation. LOEC = 1 x 105 nM RPE at MOEC = -2.57% relative to T3. |
PFDA has the potential to inhibit the function of the TH system. |
K2 Aim of the study was to assess in vitro the potential impacts of seven PFAS on TH activity using the rat pituitary tumor cell line GH3 expressing intracellular TR and responding to physiological concentration of TH by proliferation (named as T-screen). T-screen assay exposure duration 6 days. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFDA CAS No. 335-76-2. 98%. Long et al. (2013). |
Hepa1.12cR cells. 6 × 104 cells/per well 2 expts. in triplicate. Non-GL study. GLP not stated. |
1 - 1 x 105 Duration not given. |
0.02% DMSO. 60 pM TCDD. LDH. Cytotoxic at >1 x 106 nM. |
Activation / inhibition of AhR. (AhR transcriptional activation bioassay). |
Without TCDD (agonistic response). No observed effect. With TCDD (competitive response). ↓ T3-induced cell proliferation. LOEC = 1 nM. |
No effect on AhR. |
K1 No data on the number of cells plated. Aim of the study was to assess in vitro the potential impacts of seven PFAS on AhR function. The AhR transcriptional activity was determined by AhR-luciferase reporter gene bioassay using the stably transfected mouse Hepa1.1 2cR cell. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFDA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3exps. in triplicate. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 1.62 x 103 0 nM. RP = 0.019 relative to T4. TTRmutK15G. IC50 = 1.83 x 104 nM.
|
Compared to WT TTRT4 exhibited much weaker binding affinity to TTRmutK15G. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFDA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G. 1000 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G IC50 = 1.52 x 105 nM. TBGmutR381G. IC50 = 5.00 x 105 nM.
|
Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFDA CAS No. 335-76-2. ≥98%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 8.95 x 103 nM. RP = 0.007 relative to T4. |
Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Authors use the acronym PFDcA for perfluorodecanoic acid. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 16 In vitro toxicity studies for PFCAs – PFUnA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctr Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFUnA CAS No. 2058-94-8. 95%. Long et al. (2013). |
GH3 cells. 3 x 103 cells/well (96 well plate). 3 expts. in quadruplicate. Non-GL study. GLP not stated. |
1 - 1 x 105 6 days. |
0.02% EtOH. 0.5 nM T3. LDH. Cytotoxic at > 1 x 105 nM. |
Thyroid hormone-dependent cell growth (T-screen assay). |
Without T3 (agonistic response). ↓ in cell proliferation. LOEC = 1 nM. RPE at MOEC = -3.59% relative to T3. With T3 (competitive response). ↓ T3-induced cell proliferation. LOEC = 1 nM. RPE at MOEC = -2.41% relative to T3. |
PFUnA has the potential to inhibit the function of the TH system. |
K2 Aim of the study was to assess in vitro the potential impacts of seven PFAS on TH activity using the rat pituitary tumor cell line GH3 expressing intracellular TR and responding to physiological concentration of TH by proliferation (named as T-screen). T-screen assay exposure duration 6 days. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFUnA CAS No. 2058-94-8. 95%. Long et al. (2013). |
Hepa1.12cR cells. 6 × 104 cells/per well. 2 expts. in triplicate. Non-GL study. GLP not stated. |
1 - 1 x 105 Ethanol and culture medium. Duration not given. |
0.02% EtOH. 60 pM TCDD. LDH. Cytotoxic at > 1 x 106 nM (. |
Activation / inhibition of AhR. (AhR transcriptional activation bioassay). |
Without TCDD (agonistic response). ↓ AhR-tract. LOEC = 1 x 105 nM. AhR-REP No observed effect. With TCDD (antagonistic response). No observed effect.
|
No effect on AhR-tract. |
K1 No data on the number of cells plated. Aim of the study was to assess in vitro the potential impacts of seven PFAS on AhR function. The AhR transcriptional activity was determined by AhR-luciferase reporter gene bioassay using the stably transfected mouse Hepa1.1 2cR cell. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFUnA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 5.34 x 103 nM. RP = 0.006 relative to T4. TTRmutK15G. IC50 = 3.00 x 105 nM.
|
Compared to WT TTRT4 exhibited much weaker binding affinity to TTRmutK15G. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFUnA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G 1000 nM. Human mutant TBGmutR381G. 1000 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures) 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. No observed effect. TBGmutR381G. No observed effect.
|
Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFUnA CAS No. 2058-94-8. 95%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 2.16 x 104 nM. RP = 0.003 relative to T4. |
Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 17 In vitro toxicity studies for PFCAs – PFDoA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFDoA CAS No. 307-55-1. 96%. Long et al. (2013). |
GH3 cells. 3 x 103 cells/well (96 well plate). 3 expts. in quadruplicate. Non-GL study. GLP not stated. |
1 - 1 x 105. 6 days. |
0.02% EtOH. 0.5 nM T3. LDH. Cytotoxic at >1 x 105 nM. |
Thyroid hormone-dependent cell growth (T-screen assay). |
Without T3 (agonistic response). ↓ cell proliferation. LOEC = 1 nM. RPE at MOEC = -3.60% relative to T3. With T3 (competitive response). ↓ T3-induced cell proliferation. LOEC = 1 x 104 nM RPE at MOEC = -1.76% relative to T3. |
PFDoA has the potential to inhibit the function of the TH system. |
K2 Aim of the study was to assess in vitro the potential impacts of seven PFAS on TH activity using the rat pituitary tumor cell line GH3 expressing intracellular TR and responding to physiological concentration of TH by proliferation (named as T-screen). T-screen assay exposure duration 6 days. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFDoA CAS No. 307-55-1. 96%. Long et al. (2013). |
Hepa1.12cR cells. 6 × 104 cells/per well. 2 expts. in triplicate. Non-GL study. GLP not stated. |
1 - 1 x 105. Duration not given. |
0.02% EtOH. 60 pM TCDD. LDH. Cytotoxic at >1 x 106 nM (1x10-3 M). |
Activation / inhibition of AhR. (AhR transcriptional activation bioassay). |
Without TCDD (agonistic response). ↓ AhR-tract. LOEC = 1 nM ( AhR-REP = 5 x 10-6. With TCDD (antagonistic response). ↓ AhR-tract. LOEC = 1 x. 102 nM. |
PFDoA significantly induced the AhR-tact agonistic response, although no obvious concentration–response trend was observed. PFDoA affected AhR. |
K1 Aim of the study was to assess in vitro the potential impacts of seven PFAS on AhR function. The AhR transcriptional activity was determined by AhR-luciferase reporter gene bioassay using the stably transfected mouse Hepa1.1 2cR cell. No data on the number of cells plated. Study funded by the Danish Strategic Research Council and Aarhus University. |
|
PFDoA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 8.08 x 103 nM. RP = 0.004 relative to T4. TTRmutK15G. IC50 = 1.00 x 105 nM.
|
Compared to WT TTRT4 exhibited much weaker binding affinity to TTRmutK15G. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest.. |
|
PFDoA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G. 1000 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT. TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. IC50 = 280000 nM. TBGmutR381G. No observed effect.
|
Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFDoA CAS No. 307-55-1. 95%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 4.69 x 104 nM. RP = 0.001 relative to T4. |
Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 18 In vitro toxicity studies for PFCAs – PFTrDA
|
Substance / CAS no. / purity / reference |
Cell model / no. of cells / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFTrDA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 5.37 x 103 nM. RP = 0.006 relative to T4. TTRmutK15G. IC50 = 5.52 x 104 nM.
|
Compared to WT TTRT4 exhibited much weaker binding affinity to TTRmutK15G. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Authors use the acronym PFTA for perfluorotridecanoic acid. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFTrDA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G 1000 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. IC50 = 1.70 x 105 nM. RP = 2 x 10-4 relative to T4. TBGmutR378G. IC50 = 2.70 x 105 nM. TBGmutR381G. IC50 = > 4.00 x 105 nM.
|
PFTrDA and PFTeDA and were the only PFAS that bound to TBG, which is different to the pattern of binding of PFAS to TTR. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Authors use the acronym PFTA for perfluorotridecanoic acid. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
Table 19 In vitro toxicity studies for PFCAs – PFTeDA
|
Substance / CAS no. / purity / reference |
Cell model / no. of cells / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFTeDA CAS No. not given. Purity not given.
Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and mutant TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. IC50 = 6.08 x 103 nM. RP = 0.005 relative to T4. TTRmutK15G. IC50 = 1.83 x 105 nM.
|
Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. Displacement of T4 from TTR may be a concern for workers occupationally exposed to PFAS but not for the general population. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Authors use the acronym PFTdA for perfluorotetradecanoic acid. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFTeDA CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant TBGmutR381G 1000 nM. 3 expts in triplicate Non-GL study. GLP not stated. |
Human WT TBG, TBGmutR378G and TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. IC50 = 1.90 x 105 nM. RP = 2 x 10-4 relative to T4. TBGmutR378G IC50 = 2.70 x 105 nM. TBGmutR381G IC50 = > 4.00 x 105 nM.
|
PFTeDA and PFTrDA were the only PFAS that bound to TBG, which is different to the pattern of binding of PFAS to TTR. Binding affinities of TTR to PFAS were much stronger than those of TBG to the same PFAS. PFAS might bind to TTR but not TBG when ingested and enter the blood stream. |
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Authors use the acronym PFTdA for perfluorotetradecanoic acid. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
PFTeDA CAS No. 376-06-7. 96%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104 Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 2.90 x 104 nM. RP = 0.002 relative to T4. |
Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Authors use the acronym PFTdA for perfluorotetradecanoic acid. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 20 In vitro toxicity studies for PFPAs – C6 PFPA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
C6 PFPA CAS No. not given. Purity not given. Croce et al. (2019). |
WST-1 assay: FRTL-5 cells. 2 x 104 cells/well (96 well plate). 3 expts. No. of replicates not given, cAMP assay: No. cells not given (80% confluence). 3 expts. No. of replicates not given, Non-GL study GLP not stated . |
0, 1 x 10-1, 1 x 10, 1 x 102, 1 x 103, 1 x 104, 1 x 105 0 24 hr.
|
6H medium. 1 mU/L TSH (cAMP assay). WST-1 assay. No effect on cytotoxicity (data only reported in figures). |
Thyroid cell viability. TSH-stimulated cAMP production. |
No effect on inhibition of TSH-induced. cAMP production in FRTL-5 cells (data only reported in figures). |
cAMP production is not modulated by C6 PFPA . The study excludes an interference with the TSH-dependent control of thyroid function as a mechanism by which C6 PFPA would interfere with thyroid function. |
K1 Aim of the study was to investigate whether C6 PFPA could affect thyroid cell viability and/or interfere with the functional effect of TSH by investigating TSH-induced cAMP production in FRTL-5 cells. Number of replicates not given. Purity not given. No details of funding given. Authors declare no conflict of interest. |
Table 21 In vitro toxicity studies for FTOH – 6:2 FTOH, 8:2 FTOH and 10:2 FTOH
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
6:2 FTOH CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT and TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. No observed effect. TTRmutK15G. No observed effect.
|
WT TTR showed no binding towards the three fluorotelomer alcohols. No interaction between TTRmutK15G and the three fluorotelomer alcohols was observed.
|
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
6:2 FTOH CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G 1000 nM. Human mutant. TBGmutR381G 1000 nM. 3 expts. in triplicate. Non-GL study. GLP not stated. |
Human WT TBG and TBGmutR378G. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant. TBGmutR381G. 1 – 1 x 105 (est. from figures) 5 minutes. |
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. No observed effect. TBGmutR381G. No observed effect.
|
No specific comments on TBG and the three fluorotelomer alcohols.
|
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport protein TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
6:2 FTOH CAS No. 647-42-7. 98%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
8:2 FTOH CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TTR. 50 nM. Human mutant TTRmutK15G. 500 nM. 3 expts in triplicate. Non-GL study GLP not stated. |
Human WT. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant TTRmutK15G. 1 – 1 x 106 (estimated from figures). 5 minutes.
|
Competitive binding assay: NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TTR. |
WT TTR. No observed effect. TTRmutK15G No observed effect.
|
WT TTR showed no binding towards the three fluorotelomer alcohols. No interaction between TTRmutK15G and the three fluorotelomer alcohols was observed.
|
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport proteins TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
8:2 FTOH CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G. 1000 nM. 3 expts. in triplicate. Non-GL study GLP not stated. |
Human WT TBG. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant TBGmutR378G. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant. TBGmutR381G. 1 – 1 x 106 (estimated from figures) 5 minutes. |
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. No observed effect. TBGmutR381G. No observed effect.
|
No specific comments on TBG and the three fluorotelomer alcohols.
|
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport protein TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
|
8:2 FTOH CAS No. 678-39-7. 98%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND. relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
10:2 FTOH CAS No. not given. Purity not given. Ren et al. (2016). |
Human WT TBG. 50 nM. Human mutant TBGmutR378G. 1000 nM. Human mutant. TBGmutR381G. 1000 nM. 3 expts. In triplicate. Non-GL study GLP not stated. |
Human WT TBG. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant TBGmutR378G. 1 – 1 x 106 (estimated from figures). 5 minutes. Human mutant TBGmutR381G. 1 – 1 x 106 (estimated from figures). 5 minutes. |
NA. 50 nM F-T4. Cytotoxicity NA.
|
Binding potency to TBG. |
WT TBG. No observed effect. TBGmutR378G. No observed effect. TBGmutR381G. No observed effect.
|
No specific comments on TBG and the three fluorotelomer alcohols.
|
K1 Aim of the study was to investigate the binding affinities of PFAS with human TH transport protein TTR and TBG. Purity not given. Funded by the Chinese Academy of Sciences and the National Natural Science Foundation of China. Authors declared no conflicts of interest. |
Table 22 In vitro toxicity studies for FOSE – N-MeFOSA, N-EtFOSA, N-MeFOSE and N-EtFOSE
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
N-MeFOSA CAS No. 31506-32-8. ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
N-EtFOSA CAS No. 4151-50-2 ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given Non-GL study. GLP not stated. |
1 x 101 – 1 x 104 . Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
N-MeFOSE CAS No. 24448-09-7. ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
N-EtFOSE CAS No. 1691-99-2. ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 23 In vitro toxicity studies for FOSA – PFOSA, N,N-Me2FOSA
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctr Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
PFOSA CAS No. 754-91-6. ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = 6124 nM. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Authors use the acronym FOSA for perfluorooctane sulfonamide. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
|
N,N-Me2FOSA CAS No. Not given. ˃99%. Weiss et al. (2009). |
TTR. 30nM. No. of expts. and replicates not given. Non-GL study. GLP not stated. |
1 x 101 – 1 x 104. Overnight. |
NA. T4. Cytotoxicity NA. |
T4-TTR-binding. |
IC50 = ND. RP = ND relative to T4. |
RP not calculated due to IC50 falling below the laboratory detection limit. Competitive binding of PFCs to TTR, as observed for human TTR in the present study with certain PFAS, may explain altered TH levels described for PFC-exposed rats and monkeys. |
K2 Aim of the study was to investigate if PFAS compete with T4 for binding to TTR. Number of experiments and replicates not given. Study funded by EU-supported program MODELKEY and the Marie Curie Research Training Network KEYBIOEFFECTS. |
Table 24 In vitro toxicity studies for new generation substitutes - C6O4 and F-53B
|
Substance / CAS no. / purity / reference |
Test model / replicates / GL study / GLP status |
Conc. (nM) / duration |
Negative ctrl / positive ctrl Cytotoxicity |
Endpoints |
Results |
Study author conclusions |
Comments |
|
C6O4 CAS No. not given. Purity not given. Coperchini et al. (2021).
|
FRTL-5 cells. NHT cells. WST-1 assay: 2 x 104 cells/well. (96 well plate). No. of expts. and replicates not given. Annexin V‑FITC/PI assay: 1 x 104 cells/well. (24 well plate). No. of expts. and replicates not given. Cell proliferation assay: 500 cells/well (12 well plate). No. of expts. and replicates not given.
ROS production (C6O4 and FTRL5 cells only): 5 x 104 cells/well (24 well plate)
Non-GL study. GLP not stated. |
WST-1 assay: 0, 2.94 x 10-2, 2.94 x 10-1 2.94, 2.94 x 101 or 2.94 x 102. (originally reported as 0, 0.01, 0.1, 1, 10 or 100 ng/mL) 24, 48, 72, or 144 hr. Annexin V‑FITC/PI assay: 0 or 2.94 x 102. (originally reported as 0 or 100 ng/mL). 144 hr. Cell proliferation assay: 0, 2.94 x 10-2, 2.94 x 10-1 2.94, 2.94 x 101 or 2.94 x 102. (originally reported as 0, 0.01, 0.1, 1, 10 or 100 ng/mL)6 days. ROS production: 0, 2.94 x 10-2, 2.94 x 10-1 2.94, 2.94 x 101 or 2.94 x 102 0, 2.94 x 10-2, 2.94 x 10-1 2.94, 2.94 x 101 or 2.94 x 102. (originally reported as 0, 0.01, 0.1, 1, 10 or 100 ng/mL). 10, 20 or 30 minutes. |
6H medium. NA. WST-1 assay No effect on cytotoxicity (data only reported in figures). |
Cell viability. Cell death (apoptosis), late apoptotic, or necrotic cells. Cell proliferation. ROS production. |
No effect on FRTL5 or NHT cell viability at any concentration and/or time points with no induction of necrosis/apoptosis (data only reported in figures). ROS production not induced in FTRL5 cells (data only reported in figures).
|
The present in vitro study constitutes the first evaluation of the potential adverse effects of the new emerging PFAS C6O4 in cultured rat and human thyroid cells, suggesting its safety for thyroid cells in vitro. |
K2 Aim of the study was to evaluate the in vitro effects of C6O4 compared with PFOA and PFOS on primary cultures of normal human thyroid cells (NHT) in terms of cell viability, proliferation rate, and ROS production, after both short and long time of exposure. Number of experiments and replicates not given. Conversion from ng/mL based on a molecular weight of 340.05 for CAS No. 1190931-41-9. Funded by Università degli Studi di Pavia. Authors declared no conflicts of interest.
|
|
C6O4 CAS No. not given. Purity not given. De Toni et al. (2022).
|
FRTL-5 cells. Cell growth: 3-8 x 103 cells/well (96 well plate). 3 expts. in triplicate. cAMP assay: No. cells not given (80% confluence). 3 expts. in triplicate. Iodide uptake assay: No. cells not given (60% confluence) (96 well plate). 3 expts. in triplicate. Non-GL study. GLP not stated. |
0, 2.9 x 10-2, 2.9 x 10-, 2.9, 2.9 x 101 or 2.9 x 102, (originally reported as 0, 0.1, 1, 10 or 100 ng/mL). Cell growth: 24 hr. cAMP assay: 72 hr. Iodide uptake assay: 24 hr. |
Coon′s modified Ham′s F12. Medium. 1 mU/LTSH (cAMP assay) 1 mU/LTSH and NaSCN (Iodide uptake assay). MTT assay. No effect on cytotoxicity up to 2.9 x 102 nM (100 ng/mL) (data only reported in figures). |
Cell growth and membrane fluidity. Total RNA. TSH-stimulated cAMP production Iodide uptake. |
No effect on basal iodide uptake in cells in the absence of TSH stimulation. ↑ increase in iodide uptake in cells following TSH stimulation from 2.9 x 10-2 nM (0.1 ng/mL) (data only reported in figures). No effect on mean fluorescence intensity of MC540 staining (a proxy of membrane fluidity) (data only reported in figures). ↓ intracellular cAMP levels at 2.9 x 102 nM (100 ng/mL) (data only reported in figures). No effect on NIS and TPO gene expression following TSH stimulation (data only reported in figures). |
Legacy and new generation PFAS can differentially influence TSH dependent signalling pathways through the direct interaction with TSH-R. |
K2 Aim of the study was to assess the potential disrupting effect of PFAS, on FRTL-5 cells via cell function, cell toxicity, membrane biophysical properties and computational modelling of the possible interaction with TSH-Receptor. Number of replicates not given, Purity not given. Conversion from ng/mL based on a molecular weight of 340.05 for CAS No. 1190931-41-9. Study funded by Solvay Specialty Polymers Italy S.p.A. The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data, or approval of the manuscript; and decision to submit the manuscript for publication. |
|
F-53B CAS No. 73606-19-6. >99%. Deng et al. (2018). |
GH3 cells. 5000 cells/well (96 well plate). No. of expts not given. Six replicates, Non-GL study. GLP not stated. |
0, 1.75 x 104, 1.75 x 105, 1.75 x 106, 3.5 x 106, 8.75 x 106 or 1.75 x 107. (originally reported as 0, 0.01, 0.1, 1, 2, 5 or 10 mg/L) 24 hr. |
Dulbecco's Modified Eagle's Medium/Ham's F-12 medium. 1.5 µg/L T3. Cytotoxicity not assessed.
|
Thyroid hormone-dependent cell growth (T-screen assay). |
↑ relative cell proliferation from 1.75 x 104 nM (0.01 mg/L) (data only reported in figures). |
F-53B is a strong TH agonist. |
K2 Aim of the study was to investigate the thyroid disrupting potential of F-53B at environmentally relevant concentrations in rat pituitary GH3 cells (and zebrafish larvae). Cytotoxicity of F-53B to GH3 cells not assessed. Number of experiments not given. Conversion from ng/mL based on a molecular weight of 570.67 for CAS No. 73606-19-6. Study funded by National Natural Science Foundation of China, Jiangxi Province Natural Science Foundation of China, Key Program for Science and Technology Cooperation Projects of Jiangxi, China, Jiangxi Science and Technology Project, Key Program of Jiangxi Academy of Sciences and Program of State Administration of Foreign Experts Affairs. |