6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

The study is given a K4 score because it concerns a newly developed in vitro screening method that was not yet internationally validated.

Data source

Reference

Reference Type:

publication

Title:

In vitro screening for chemical inhibition of the iodide recycling enzyme, iodotyrosine deiodinase

Author:

Jennifer H. Olker, Joseph J. Korte, Jeffrey S. Denny, Jonathan T. Haselman, Phillip C. Hartig, Mary C. Cardon, Michael W. Hornung, Sigmund J. Degitz

Year:

2021

Bibliographic source:

Toxicology in Vitro (2021), 71, p. 105073.

Materials and methods

Principles of method if other than guideline:

It concerns an in vitro assay to assess IYD (iodotyrosine deiodinase) inhibition. IYD is an enzyme that maintains adequate iodide concentrations for thyroid hormone (TH) synthesis.
The principle of the assay is based on a colorimetric readout that measures the release of iodide from the substrate.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: endocrine effects

Test material

Test material information

1

Reference substance name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

EC Number:

204-327-1

EC Name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Cas Number:

119-47-1

Molecular formula:

C23H32O2

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol

Administration / exposure

Doses / concentrations

Dose / conc.:

200 other: µM

Details on study design:

Assay details:
- Non-radioactive method in a 96-well plate with a colorimetric readout that measures release of iodide from the substrate.
- IYD enzyme: recombinant human IYD enzyme produced in a baculovirus system with insect cells. The sequence encoding the full length IYD gene was obtained from GenBank BC056253.1 open reading frame.
- Substrate: monoiodotyrosine (MIT)
- Reducing agent: β-nicotinamide adenine dinucleotide 2′-phosphate (reduced form) (NADPH)
- Positive control: 3-nitro-L-tyrosine (MNT)
- Negative (solvent) controls: 0.05M NaOH and DMSO
- Test concentration: 200 µM (single concentration testing). After the single concentration test, an additional concentration-response screening was performed with concentrations ranging from 0.032 to 200 µM.
- IYD-containing cell preparations typically contained ca. 1.8 mg/mL total protein (as per Bradford assay using Bovine Serum Albumin (BSA)). The preparations were diluted 2 to 3-fold with water. The diluted cell suspensions were briefly homogenized by sonication, and diluted to contain an amount of activity that would produce the optimum iodide release for a 15-min. Sandell-Kolthoff (SK) reaction to result net change in absorbance of ca. 0.6 absorbance units after a 3h chemical inhibition assay incubation. A typical assay contained ca. 25 µg cell lysate protein per well. Under these conditions the specific activity was determined to be ca. 3 pmoles iodide per hour per µg of protein.

Chemical screening:
- Test chemicals were supplied in 96-well plates with one chemical per sample per well.
- Target stock concentration in the well: 20 mM in DMSO.
- Each chemical source plate was tested on 3 individual assay plates on the same day, i.e. n = 3 data points for each chemical.
- Data were normalized to percent of control with solvents (DMSO, NaOH) representing maximal enzyme activity (0% inhibition), and 200 µM MNT as no enzyme activity (100% inhibition). Chemicals that produced inhibition >= 20% were considered potential IYD inhibitors.

Results and discussion

Details on results:

Median inhibition observed at 200 µM limit test: 72%.
The concentration-response screening assessment yielded the following values:
- IC20: 7.7 µM
- IC50: 28.9 µM

Applicant's summary and conclusion

Conclusions:

6,6'-Di-tert-butyl-2,2'-methylenedi-p-cresol was found to reduce iodotyrosine deiodinase (IYD) by 72% at a concentration of 200 µM in vitro. As this result is obtained through a newly developed in vitro assay that is not yet internationally validated, the relevance and reliability of this finding is not yet fully established.