Abstract

Thermal receipt papers have undergone rapid reformulation in response to regulatory pressure on bisphenol A (BPA), yet there is limited information on the overall endocrine activity of alternative products. This work evaluates the endocrine-disrupting potential of three commercially available, unprinted thermal papers—BPA-based, “BPA-free,” and “non-bisphenol”—using effect-directed in-vitro assays that capture the combined impact of all leachable components rather than isolated chemicals. Shredded papers were extracted in 10% ethanol in culture medium and applied to GH3 rat pituitary cells and H295R human adrenocortical carcinoma cells at concentrations up to 0.15 mg putative color developer per g paper equivalent. In GH3 cells, all three extracts induced a dose-dependent down-regulation of Tshβ mRNA, with BPA and “BPA-free” papers producing the strongest effects, while thyroid receptor (Trα, Trβ) and deiodinase (Dio1, Dio2) transcripts were less consistently affected. The same BPA and “BPA-free” extracts significantly reduced Fsh, Erα, and Erβ expression, whereas the non-bisphenol paper showed weaker changes confined mainly to Fsh. In H295R cells, all three extracts increased 17β-estradiol (E2) secretion at non-cytotoxic concentrations, without altering testosterone levels or transcription of CYP19A1, CYP17A1, or StAR, suggesting interference with estrogen metabolism rather than de novo synthesis. Across both models, “BPA-free” and “non-bisphenol” papers exhibited measurable endocrine activity, albeit generally lower than the BPA paper. These findings demonstrate that marketing claims alone do not guarantee endocrine safety and underline the importance of effect-based screening for entire thermal paper formulations, not just single target analytes.

Keywords

Thermal paper; BPA-free; endocrine disruption; GH3 cells; H295R assay; Tshβ; Fsh; estradiol; mixture toxicity; regrettable substitution.

1. Introduction

Thermal receipt paper is a small, everyday product that sits at the intersection of analytical chemistry, toxicology and consumer safety. For decades, bisphenol A (BPA) served as the dominant color developer in thermal coatings, enabling rapid, inkless printing for point-of-sale receipts, tickets and labels. As concerns about BPA’s endocrine-disrupting effects accumulated—particularly its estrogenic, thyroid and metabolic impacts—regulators, retailers and paper manufacturers began to push for BPA reduction and replacement.

A large body of work has since quantified BPA and its analogues such as bisphenol S (BPS) in receipts and other thermal papers, and has evaluated the effects of these pure chemicals in cell and animal models. However, thermal papers are mixtures, not pure substances. Their coatings contain color developers, leuco dyes, pigments, binders, sensitizers, stabilizers and other proprietary additives. Focusing exclusively on BPA or BPS may underestimate the hazard if other formulation components contribute to endocrine activity, or if substitutes introduced under “BPA-free” and “non-bisphenol” claims carry their own biological effects.

Effect-based bioassays offer a complementary approach: rather than testing isolated compounds, one can extract real-world materials and observe how the combined leachate perturbs hormone-sensitive systems. The OECD-endorsed H295R assay, for example, is widely used to screen substances that alter steroid hormone synthesis, while GH3 pituitary cells are a classic model for thyroid-axis modulation.

The present work synthesizes and structures the findings of a thesis that applied such effect-directed testing to three categories of unprinted thermal receipt paper: a conventional BPA-based paper, a “BPA-free” paper, and a “non-bisphenol” paper. The central questions were:

1. Do whole extracts from these papers disrupt markers of pituitary thyroid and gonadal regulation in GH3 cells?
2. Do they alter estradiol and testosterone production in H295R cells?
3. Are “BPA-free” and “non-bisphenol” papers demonstrably safer than conventional BPA-based paper in these in-vitro systems?

By framing the study around whole-formulation extracts, the work reflects more closely the actual exposure scenario for workers and consumers who handle receipts, and tests whether substitution strategies have genuinely reduced endocrine activity or merely shifted it to less familiar chemicals.

2. Materials and Methods

2.1 Thermal Papers and Extraction

Three unprinted thermal receipt papers were purchased from an online marketplace:

1. A conventional BPA-based paper.
2. A “BPA-free” paper, labelled as such by the supplier.
3. A “non-bisphenol” paper, marketed as not containing bisphenol developers.

No independent chemical characterization of the color developers or additives was conducted; the focus was on the net biological activity of the leachable fraction.

For each paper type, sheets were shredded and weighed. Portions of 1.5 g shredded paper were extracted with 10 mL of cell culture medium containing 10% (v/v) ethanol. Extraction involved vortex mixing and 15 min sonication, intended to liberate a broad spectrum of soluble and semi-soluble components. After centrifugation and removal of particulates, the supernatants were used as stock extracts.

Following the convention of Mendum et al. (2011), the color-developer content was assumed to be 1% of the paper weight. On this basis, exposure levels were expressed as mg developer per g receipt paper equivalent in the culture system. Test concentrations were:

• 0 (vehicle control, 0.1% ethanol in medium)
• 0.0015 mg/g
• 0.005 mg/g
• 0.015 mg/g
• 0.05 mg/g
• 0.15 mg/g

These levels span several orders of magnitude below nominal developer content, capturing both low- and high-range effects.

2.2 GH3 Pituitary Cell Assay

Rat GH3 pituitary cells, sensitive to thyroid hormone and involved in pituitary control of both thyroid and gonadal axes, were used to examine gene expression responses.

Cells were exposed to the paper extracts for 48 h. Triiodothyronine (T3) served as a positive control at 0.0065, 0.065 and 0.65 µg/L. After exposure, RNA was isolated and reverse-transcribed, and mRNA levels were quantified by qPCR for:

• Thyroid-axis markers
  • Tshβ (thyroid-stimulating hormone β subunit)
  • Trα, Trβ (thyroid hormone receptors α and β)
  • Dio1, Dio2 (deiodinases 1 and 2)
• Pituitary gonadal-axis markers
  • Fsh (follicle-stimulating hormone)
  • Erα, Erβ (estrogen receptors α and β)

Expression was normalized to housekeeping genes and reported as fold-change relative to vehicle controls.

2.3 H295R Steroidogenesis Assay

Human H295R adrenocortical carcinoma cells, the basis of the OECD steroidogenesis assay, were used to evaluate changes in sex steroid production.

Cells were exposed for 48 h to the same concentration range of paper extracts. Forskolin served as a positive control for steroidogenic induction. At the end of exposure, culture medium was collected, and concentrations of:

• 17β-estradiol (E2)
• Testosterone (T)

were quantified by immunoassay or LC-MS/MS, depending on the laboratory protocol. In parallel, RNA was extracted to measure expression of key steroidogenic genes:

• CYP19A1 (aromatase)
• CYP17A1
• StAR (steroidogenic acute regulatory protein)

Again, expression was expressed as fold-change versus vehicle controls.

2.4 Cytotoxicity Assessment

To distinguish true endocrine-mediated changes from nonspecific toxicity, cytotoxicity was first assessed by WST-1 assay in both GH3 and H295R cells across the full concentration range. Endpoints showing strong reductions in viability were excluded from mechanistic interpretation, and emphasis was placed on concentrations at or below 0.05 mg/g, where cell viability remained acceptable.

3. Results

3.1 Cytotoxicity Profiles

In both GH3 and H295R cells, T3 and forskolin behaved as expected, with T3 modestly stimulating GH3 proliferation and forskolin driving steroidogenic activity without overt cytotoxicity.

For the paper extracts:

• BPA-based and “BPA-free” papers showed reduced cell viability at the highest tested concentration (0.15 mg developer/g paper) in both cell lines.
• The non-bisphenol paper did not cause pronounced cytotoxicity or hyper-proliferation across the tested range.

Subsequent analyses of gene expression and steroidogenesis therefore focused primarily on ≤0.05 mg/g concentrations, representing non-cytotoxic or minimally cytotoxic exposure levels.

3.2 Thyroid-Axis Markers in GH3 Cells

3.2.1 Tshβ

The most consistent and striking finding was the behavior of Tshβ mRNA:

• All three extracts produced a dose-dependent down-regulation of Tshβ.
• For the BPA-based and “BPA-free” papers, suppression of Tshβ was statistically significant across multiple concentrations.
• The non-bisphenol paper showed a similar downward trend, but the magnitude of change was smaller and sometimes failed to reach significance.

The direction of effect closely paralleled that of T3, which reduced Tshβ via negative feedback, suggesting that components within the extracts can mimic or disrupt thyroid feedback mechanisms at the pituitary level.

3.2.2 Trα, Trβ, Dio1, Dio2

In contrast, thyroid receptor transcripts Trα and Trβ and deiodinases Dio1 and Dio2 were much less affected:

• High-dose T3 reduced Trα and Trβ, as expected, confirming assay sensitivity.
• None of the three paper extracts produced robust, consistent changes in Trα or Trβ.
• Dio1 showed a significant decrease only in cells exposed to the BPA-paper extract; Dio2 expression remained largely unchanged.

These patterns suggest that the extracts influence the central pituitary thyroid regulator (Tshβ) more strongly than receptor or deiodinase gene expression, pointing to non-classical modes of interference with the thyroid axis.

3.3 Pituitary Gonadal-Axis Markers in GH3 Cells

For the reproductive axis markers:

• BPA and “BPA-free” paper extracts significantly down-regulated Fsh, Erα and Erβ mRNA in a concentration-dependent manner.
• The non-bisphenol paper showed a weaker pattern: Fsh tended downward, with occasional significance, but Erα and Erβ remained largely unchanged.

Given literature evidence that estradiol can reduce FSH release and down-regulate Erβ in pituitary cells, these results are consistent with weak estrogenic influences within the BPA and “BPA-free” extracts, extending endocrine disturbance beyond the thyroid-axis to gonadotropic signaling.

3.4 Steroid Hormone Production in H295R Cells

After 48 h exposure, all three extracts impacted 17β-estradiol (E2) production:

• E2 levels increased significantly at the highest non-cytotoxic concentrations for each paper type:
  • BPA paper: pronounced E2 elevation at 0.05 mg/g.
  • “BPA-free” paper: moderate E2 increase at 0.05 mg/g.
  • Non-bisphenol paper: smaller E2 increase observed at 0.15 mg/g, roughly half the amplitude seen with the BPA paper at 0.05 mg/g.
• Testosterone levels did not change significantly across treatments.

Consequently, the E2/T ratio shifted towards higher estrogenicity in the culture medium for all three paper types, with BPA > “BPA-free” > non-bisphenol in apparent potency.

3.5 Steroidogenic Gene Expression

Despite the observed changes in estradiol production:

• CYP19A1 (aromatase) expression did not show a consistent pattern of up-regulation or down-regulation in response to any of the extracts.
• CYP17A1 and StAR also remained largely unaffected at the transcriptional level.

The disconnect between higher E2 concentrations and stable CYP19A1, CYP17A1 and StAR mRNA suggests that estradiol elevation is unlikely to result from enhanced de-novo synthesis. Instead, the data align with previous reports in which BPA increased E2 concentrations by inhibiting estrogen metabolism or clearance rather than by increasing its production, implicating pathways such as sulfotransferases or UDP-glucuronosyltransferases that were not directly measured in this work.

4. Discussion

4.1 Endocrine Activity Across All Paper Types

Collectively, the GH3 and H295R data give a consistent message: all three thermal papers—BPA-based, “BPA-free,” and “non-bisphenol”—exhibited measurable endocrine activity in vitro. Even when cytotoxicity was excluded by focusing on lower concentrations, the extracts altered key markers:

• Central thyroid regulation via Tshβ suppression.
• Pituitary gonad-axis markers (Fsh, Erα, Erβ) for BPA and “BPA-free” receipts.
• Estradiol secretion in a human steroidogenic cell line for all three paper types.

The non-bisphenol paper generally showed lower potency, but it was not biologically inert. Its extract still suppressed Tshβ and elevated E2 at the top non-toxic concentration, indicating that the formulation contains one or more components capable of modulating endocrine-sensitive pathways.

4.2 “BPA-Free” and “Non-Bisphenol” Do Not Equal “Endocrine-Safe”

A central implication is that common marketing labels—“BPA-free” and even “non-bisphenol”—do not guarantee the absence of endocrine-active substances. Given the lack of chemical analysis in this study, one cannot identify which components are responsible—whether alternative bisphenols (e.g. BPS, BPF, BPS-MAE), non-phenolic developers (e.g. Pergafast-201, D-8, NKK-1304), or other additives and process chemicals.

From a risk communication and product-stewardship standpoint, this supports a more cautious message: removing BPA is necessary but not sufficient. Without full characterization and effect-based screening of the replacement formulations, there is a real possibility of “regrettable substitution”, where new chemicals carry similar or novel endocrine activities.

4.3 Mechanistic Considerations

The pattern of responses offers some mechanistic clues:

• Thyroid axis: Strong and consistent Tshβ suppression, with relatively modest effects on Tr and Dio genes, is compatible with indirect disruption of central thyroid regulation rather than simple agonism or antagonism at the thyroid hormone receptor. Bisphenols are known to interact with multiple nuclear receptors and co-regulators, and to affect deiodinase activity and transporters, so multiple overlapping mechanisms are plausible.
• Gonad axis: Down-regulation of Fsh, Erα and Erβ by BPA and “BPA-free” papers aligns with their general estrogen-mimicking behavior. Reduced expression of estrogen receptors may reflect feedback adaptation to sustained low-level estrogenic signaling.
• Steroidogenesis: The increase in estradiol without parallel changes in CYP19A1, CYP17A1 or StAR points towards altered estrogen metabolism, possibly via inhibited conjugation or excretion pathways, a mechanism suggested in earlier BPA studies. This would mean that even low aromatase activity could generate higher steady-state E2 levels if clearance is compromised.

These mechanistic impressions remain hypothesis-generating; targeted assays for metabolic enzymes and receptor binding would be required to confirm them.

4.4 Strengths and Limitations of the Effect-Based Approach

The primary strength of the study lies in its mixture-oriented, effect-directed design. By extracting real-world papers, the tests capture:

• Co-exposure to multiple coating ingredients.
• Potential synergy or antagonism within mixtures.
• Bioactive components that would be missed by targeted BPA/BPS analytics.

The twin-assay strategy (GH3 + H295R) adds value by covering both thyroid and reproductive axes, integrating gene expression with hormone output and cytotoxicity checks.

At the same time, important limitations must be acknowledged:

• Only three paper types were examined; they cannot represent the full variability of thermal papers on the market.
• No chemical analysis was undertaken, preventing assignment of effects to specific substances or quantification of bisphenols and their levels.
• The developer content was assumed, not measured, and real formulations may deviate from the 1% reference value.
• The work is in-vitro, short-term and not translated into internal doses or population-level risk metrics.

Thus, the findings are best interpreted as hazard identification and comparative screening, not as a complete risk assessment.

5. Conclusion

This study provides a structured view of the endocrine activity of three categories of thermal receipt papers when tested as whole-formulation extracts in hormone-sensitive cell models. Several clear conclusions emerge:

1. BPA-based thermal paper exerts robust endocrine-disrupting effects on thyroid and reproductive markers in GH3 cells and enhances estradiol production in H295R cells.
2. “BPA-free” thermal paper shows qualitatively similar patterns—down-regulation of Tshβ, Fsh, Erα, Erβ, and increased estradiol—although typically with lower potency than the BPA paper.
3. “Non-bisphenol” paper is less potent but still capable of suppressing Tshβ and elevating estradiol at higher, non-cytotoxic concentrations, indicating that entirely different developer systems can also perturb endocrine endpoints.
4. The label “BPA-free” or “non-bisphenol” therefore cannot be equated with “endocrine-safe.” Systematic effect-based screening and, where necessary, in-vivo studies are essential to validate the safety of replacement formulations.
5. For regulators, retailers and manufacturers, the message is that substitution must be guided by hazard data, not just by the absence of a single chemical name on the specification sheet.

As industry continues to move away from BPA, the challenge is not only to eliminate a well-known endocrine disruptor, but to design and adopt thermal paper systems that have been demonstrably optimized for minimal endocrine activity as mixtures, underpinned by transparent analytical and toxicological evaluation.

Last Updated on 12/12/2025 by Jota Machinery