The primary investigator (Y.L.L.) reviewed a list of 39 tattoo ink companies that manufactured and sold tattoo inks in the United States; this list was generated from a previous study of tattoo ink pigments.⁹ To minimize counterfeit products, tattoo inks were excluded if they (1) could not be purchased through a US online tattoo product wholesaler or through respective individual Web sites, (2) were available exclusively through Amazon.com, or (3) were discontinued.

A dermatologist with expertise in tattoo use (W.L.) categorized the brands of tattoo inks into 3 different groups (common, uncommon, and rare) based on the popularity and prevalence of use by US tattoo artists (Table 1). Five inks of primary colors (including red, yellow, blue, white, and black) were purchased for all brands. Three inks of secondary colors (including orange, green, and purple) were also purchased for common brands. A few brands including “One Ink,” “Kokkai,” and “Cheyenne” had limited colors; in these cases, all colors available were purchased. All tattoo inks used in this study were purchased from online tattoo wholesale/retail vendors or their respective websites.

The label for each tattoo ink was examined for the presence of declared formaldehyde/formaldehyde releasers (eg, bronopol, DMDM hydantoin, diazolidinyl urea). If no ingredients were available on the label, the online Material Safety Data Sheet, if available, was examined and reviewed.

All chromotropic acid experiments were conducted at room temperature using standard CAM procedures as described in previous studies.²⁴ Half of a milliliter of tattoo ink sample was used for each experiment. For each chromotropic acid experiment, 0.5 mL of 0.1% formaldehyde solution was used as a positive control, and distilled water was used as a negative control. Each ink sample was tested twice on separate days. Samples that provided conflicting results from the first 2 experiments were tested a third time.

After 48 hours of incubation, each vial of chromotropic acid solution with either tattoo ink samples or control solutions was placed in a Beckman DU 640 UV-VIS spectrophotometer (Fig. 1A). The chromotropic acid solution from the negative control served as blank for each absorbance reading.²⁴ Absorbances of 570- and 573-nm wavelength of light were used for optimal formaldehyde detection.²⁵

To identify the limit of detection (LOD) and set the threshold of positivity, a dose-dependent formaldehyde titration curve was performed 3 independent times. Formaldehyde solutions from 2.5 to 80 ppm in concentration with 2-fold dilutions (Fig. 2) were used to form a standard curve. First, an 80-ppm (0.008%) formaldehyde solution was made from a 0.1% formaldehyde solution and then serially diluted to create standard solutions. Each standard formaldehyde solution was incubated with chromotropic acid solution per protocol and then read using the spectrophotometer (Fig. 1B). The LOD for the spectrometer in identifying formaldehyde was determined by 3.3 times the standard deviation of the regression line divided by the slope. Each regression line was generated using data of concentration ranges of 0 to 5 ppm or 0 to 10 ppm, whichever generated a line with the highest R². Data of higher concentrations (20–80 ppm) were excluded because they were outside the linear range for spectrophotometric measurement. The average LOD of 4 standard titration sets was used as the threshold for positivity for all ink samples tested. Absorbance of 2.5-ppm formaldehyde solution was determined as the lowest relevant concentration per previous guidelines.²⁴

Of the 127 inks, 93 inks (73%) had listed ingredients, and 34 inks (27%) did not. Of those with ingredients, 80 tattoo inks did not declare formaldehyde or other formaldehyde-releasing chemicals as ingredients, whereas 13 inks listed DMDM hydantoin. The 13 DMDM-hydantoin–containing tattoo inks were from 2 brands, Millennium Moms Tattoo Ink and World Famous.

The LOD of the spectrophotometer was calculated to be 2.47 ppm. The average absorbance for a 2.5-ppm formaldehyde solution was determined to be 0.014 AU when read with 570-nm light and 0.013 when read with 573-nm light. On the basis of this determined threshold of positivity, 93 of the 127 tested tattoo inks (73%) released greater than or equal to 2.5 ppm of gaseous formaldehyde; 34 of the 127 tested tattoo inks (27%) released less than 2.5 ppm of gaseous formaldehyde (Table 2). All inks with DMDM hydantoin were positive for formaldehyde release using CAM.

This study has notable limitations. Most importantly, we chose a convenience sample of predispersed tattoo ink brands manufactured and sold in the United States, based on common, uncommon, and rare commercial brands, which spanned a wide variety of tattoo ink manufacturers. We selectively tested the more commonly used primary and secondary colors. Tattoo inks were excluded from this study to avoid counterfeit inks and to ensure purchase from reliable sources that tattoo parlors would be likely to use for bulk purchases. Testing potentially counterfeit inks from less reliable sources may have yielded additional products containing formaldehyde and may represent a direction for future studies.