Registration Dossier

Administrative data

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-standard in vitro assays, but methodology used and results well described.

Data source

Reference
Reference Type:
publication
Title:
The in vitro estrogenic activities of polyfluorinated iodine alkanes
Author:
Wang C., Wang T., Liu W., Zhou Q., Liu J., Zhang A., Zhao B., and Jiang G.
Year:
2012
Bibliographic source:
Environ. Health Perspect. 120:119-125

Materials and methods

Principles of method if other than guideline:
3 in vitro assays were used:
E-screen assay: in response to estrogen receptor-alpha agonists, the mitotic effect leads to the proliferation of MCF-7BUS cells.
MVLN assay: estrogen receptor agonists can induce the production of luciferase in the MVLN cell line that have been stably transfected with the luciferase reporter gene and estrogen-responsive element derived from Xenopus vitellogenin A2 gene.
Estrogen-responsive gene: gene expression in MCF-7BUS cells analysed by quantitative RT-PCR
GLP compliance:
not specified
Type of method:
in vitro
Endpoint addressed:
other: estrogenic activity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test materials (as cited in study report):
Dodecafluoro-1,6-diiodohexane (PFHxDI, C-6), 97% pure
Octafluoro-1,4-diiodobutane PFBDI, C-4, 97% pure
Hexadecafluoro-1,8-diiodooctane, PFODI, C-8, 98% pure

and 10 other test compounds: fluorinated iodine alkanes, fluorinated telomer iodides, fluorinated diiodine alkanes, as well as perfluorooctane, 1-bromoperfluorooctane, perfluorooctanoic acid and 1-Iodohexane..

- Physical state: not reported
- Analytical purity: 97%
- source: Alfa Aesar (Ward Hill, MA, USA)

Administration / exposure

Route of administration:
other: in vitro assays with cultured cells
Vehicle:
ethanol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dodecafluoro-1,6-Diiodohexane (PFHxDi) and octafluoro-1,4-diiodobutane were dissolved in ethanol. The stock solution was stored at -20°C.

CELL CULTURES:
- cell types:
Human MCF-7BUS breast adenocarcinoma cells for E-screen assay
MVLN cells
- cell cultures: cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F-12 with 10% fetal bovine serum, supplemented with 100 U/ml streptomycin-penicillin, 2 mM L-glutamine, and 1% insulin-transferrin-selenium. Cells were cultured in 100-mm culture dishes in humidified atmosphere, 5% CO2, at 37°C.

E-screen assay:
Performed according to protocol by Soto et al. (1995) [Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO. 1995. The E-SCREEN assay as a tool to identify estrogens—an update on estrogenic environmental-pollutants. Environ Health Perspect 103(suppl 7):113–122]
MCF-7BUS cells were plated in 96-well plates at the density of 3000 cells/well. Before each experiment cells were starved in steroid‑free (SF) medium for 48 hr to minimize the basal hormonal activity during assays.
SF medium: phenol red–free DMEM/F-12 supplemented with 5% dextran-charcoal-treated fetal bovine serum, 100 U/mL streptomycin-penicillin, and 2 mM l‑glutamine.
- Concentration range of test substance: serial dilutions from 1 nM to 100 μM in SF medium.
- Positive control: concentration range of 0.01–200 pM 17β-estradiol (E2; 99% pure).
- Proliferation/viability was assessed after 6-day exposure period using WST-1 kit (measures the enzymatic cleavage of the tetrazolium salt of WST-1 to formazan by cellular mitochondrial dehydrogenases in viable cells). Absorbance of the WST‑1 solution was detected at 450 nm, with the reference wave length at 690 nm. The cell proliferation was then calculated from the solvent control (0.1% ethanol)-corrected absorbance and expressed as the percentage of maximal absorbance of the positive control.
Three replicates in each experiment.

MVLN assay:
MVLN cells were seeded in the interior 60-wells of 96-well plates at the density of 70000 cells/well, starved in SF medium for 48-h, and exposed to the test compounds for 2 days.
- Concentration range of test substance: 0.1-100µM
- Positive control: concentration range of 0.5 pM–1 nM 17β-estradiol (E2; 99% pure).
- Luciferase activity measured with the LucLite kit, with a microplate reader (integration of luminescence signal for 10s).
- Total protein assay: Bradford test.
The results are expressed as relative luminscence unit/µg of protein.
The positive control (corrected for solvent control, 0.1–0.2% ethanol) defines the maximal induction as 100%, and the responses of other chemicals were converted to a percentage of the maximum level.
Cytotoxicity: checked in parallel using WST‑1 kit, and regurlary observed under microscope to identify the exposure concentration range.
Three replicates in each experiment.

Gene expression by reverse-transcription polymerase chain reaction (RT-PCR):
MCF-7BUS cells were plated in 6-well plates at the density of 1.10E6 cells/well, starved in SF medium for 48-h, and exposed to the test compounds for 2 days. Total RNA was isolated according to standard method, and quantified by reading at 260 nm, and quality was checked by 260:280 nm ratios obtained between 1.8 and 2.0.
Gene expression was quantified using 2-step quantitative RT-PCR.
The primer sequences used were:
- β‑actin (internal gene used for reference),
- Early Growth Response protein 3 (EGR 3). Estrogen-receptor-mediated estrogen-inducing gene. Early growth response family.
- Trefoil factor 1 (TFF1; pS2). Gene involved in cell proliferation. Also a biomarker responding to estrogens.
Expression of each target gene was normalised with β‑actin used as reference. The fold-change of the target genes was analysed by the 2-∆∆Ct method (reference: Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25(4):402–408)
Analytical verification of doses or concentrations:
not specified

Results and discussion

Details on results:
Estrogenic activity in MCF-7BUS cells in E-screen assay (results in table 1):
A full concentration-response curve was observed for all 3 diiodofluorinated alkanes with the following intensity:
Dodecafluoro-1,6-diiodohexane (PFHxDI, C-6) > Hexadecafluoro-1,8-diiodooctane, PFODI, C-8 > Octafluoro-1,4-diiodobutane PFBDI, C-4.

Proliferation results:
PFBDI (C-4) EC50= 1.45 µM > PFHxDI (C-6) EC50= 7.5 nM > PFODI (C-8) EC50= 43.3 nM


Transactivation in MVLN cells (results in Table 2):
No significant cytotoxicity was observed within the concentration ranges and no cytotoxicity was observed by microscopic examination.
Because there was no maximum induction compared to estradiol, the relative potency ratios calculated based on EC20 were considered more robust (according to Villeuneve DL, Blankenship AL, Giesy JP. 2000. Derivation and application of relative potency estimates based on in vitro bioassay results. Environ Toxicol Chem 19(11):2835–2843).
There was a dose-dependent induction of luciferase activity for PFBDI (C-4), PFODI (C-8), and PFHxDI (C-6) and effects related to the carbon chain length.
Estrogenic activity was higher with PFHxDI (C-6). Effects were similar to those in the E-screen asssay.

Comparison with non-fluorinated structures and non-iodinated structures:
The non-fluorinated hydrocarbon 1-iodohexane did not show estrogenic effects in either assays.
Several 8-carbon fluorinated compounds without iodine substitution did not show estrogenic effects in either assays.
The association of polyfluorinated alkyl chain and iodine substitution are critical for the estrogenic activity of polyfluorinated iodine alkane derivatives.

Coexposure assay of polyfluorinated diiodine alkanes and 4-hydroxytamoxifen (OHT):
OHT is a strong estrogen agonist in the mammary gland. Coexposure of OHT with the polyfluorinated iodine alkane resulted in a strong reduction of luciferase activity. The iodinated compounds are thus interacting with the estrogenic receptor.

Estrogen receptor expression (results in Table 3):
All 3 diiodinated derivatives induced the estrogen-responsive genes.
No significant gene expression induction was observed with the non-fluorinated derivative 1-iodohexane or non-iodinated fluorinated derivatives.

Additional results in the publication indicated that the activities were also higher with the compounds with 2 iodine substitutions compared to one.

Any other information on results incl. tables

Table 1 – Maximum induction and effective concentration in the E-screen assay

compound

Maximum induction (%)a

EC50

Relative potencyb

17β-estradiol (reference)

100

3.12 pM

1

FBDI (C-4)

97

1.45 µM

2.2 x 10-6

PFHxDI (C-6)

100

7.5 nM

4.2 x 10-4

PFODI (C-8)

105

43.3 nM

7.2 x 10-5

a Percentage of the maximum proliferation effects of tested compounds to that of 17β-estradiol.

b Ratio of EC50 of 17β-estradiol to that of test compounds

Relative potency in E-screen assay: PFHxDI (C-6) > PFODI (C-8) > FBDI (C-4)

(6 orders of magnitude below that of estradiol)

Table 2 – Maximum induction and effective concentration in the MVLN assay

compound

Maximum induction (%)a

EC50

EC20

Relative potencyb

17β-estradiol (reference)

100

16.4 pM

4.14 pM

1

FBDI (C-4)

21

29.6 µM

13.8 µM

0.3 x 10-6

PFHxDI (C-6)

73

1.13 µM

0.38 µM

11 x 10-6

PFODI (C-8)

38

2.87 µM

1.07 µM

3.8 x 10-6

a Percentage of the maximum induction effects of tested compounds to that of 17β-estradiol.

b Ratio of EC20 of 17β-estradiol to that of test compounds, because there was no maximum induction compared to estradiol

Relative potency in MVLN assay: PFHxDI (C-6) > PFODI (C-8) > FBDI (C-4)

(5 to 6 orders of magnitude below that of estradiol)

In both assays, the highest activity was obtained with the 6 carbon chain length.

Table 3 – Up-regulation of estrogen-responsive genes

compound

concentration

TFF1*

EGR3*

17β-estradiol

100 pM

8.7

9.2

PFBDI (C-4)

50 µM

2.7

2.4

PFHxDI (C-6)

20 µM

5.7

9.1

PFODI (C-8)

40 µM

8.5

11.2

* Fold increase above control (0.1% ethanol) following 48-hr treatment

 

Applicant's summary and conclusion

Conclusions:
Three polyfluorinated diiodine alkanes Dodecafluoro-1,6-diiodohexane (PFHxDI, C-6), Octafluoro-1,4-diiodobutane (PFBDI, C-4), and Hexadecafluoro-1,8-diiodooctane (PFODI, C-8) displayed estrogenic activity in 2 in vitro assays, and up-regulation of the estrogen-responsive genes TFF1 and EGR3 indicated that the estrogenic activity could be mediated through activation of the estrogen receptor.
The relative estrogenic potencies were dependent on the carbon chain length (maximum activity with 6 carbons) and iodine substitution.