The mechanism by which copper accelerates color formation is proposed to be accelera- tion of the initial primary intermediate to diamine reaction, as shown in Figure 3. This oxidation with hydrogen peroxide in a “clean” solution with no added redox metals is kinetically a slow step that proceeds via two one-electron oxidations. Ad ded redox metals accelerate the initial formation of semiquinone radical cation, which then rapidly forms the diimine. HAIR EXPERIMENTS Hair experime nts were carried out to determine how copper acceleration of dye formation impacts color uptake on hair. Previously colored hair was fi rst rinsed in tap water contain- ing 0.06–0.08 μg/g of copper to create sets of 12 tresses with four different copper levels (Ta ble I). Water hardness was medium (~9 US gpg), and no signifi cant differe nces in calcium and magnesium were measured between tresses. These copper levels are within the range of levels seen in consumers’ hair from previously reported hair harvesting (4). Each set of tresses was then co lored with three different shades [intense medium blonde/ red (77/44), medium brown/violet (4/6), and intensive red (66/46)] to asses the impact of copper levels in hair on color formation. The shades were chosen as ones that have the same dye primary intermediates and coupler combinations used in the solution testing. Figure 4A–C shows L*, a*, and b* values for initial color results. In all shades, a signifi - cant difference is measured in color readings as a function of copper levels. A higher L* value is seen with higher copper levels, indicating the shade is lighter due to lower dye Figure 2. Change in L at 7 min versus initial for PPD–AHT, HDAP–AHT, and PPD–Res dye couples. REDOX METALS IN COLOR FORMATION IN A HAIR COLORANT 273

levels. In addition, a* and b* values are different, indicating the visual color is also a dif- ferent tone, and this was also observed. In all cases, at lower copper levels in hair the fi nal color was darker and more vibrant, e.g., redder for shade 77/44 and more violet for shade 4/6. The L*a*b* color space is not l inearly correlated with dye intensity, so one shade was used to more accur ately correlate copper level in hair before coloring with fi nal dye up- take. Shade 77/44 was chosen as it contains only two dye precursors—HDAP and AHT— and the predominant red color formed has a peak at 477 nm (ƐEtOH = 18,900 M-1.cm-1). This is the formed dye: (Z)-5 -amino-4-((2-amino-1-(2-hydroxyethyl)-1H-pyrrol-3-yl) imino)-2-methylcyclohexa- 2,5-dien-1-one (Figure 5). A 2:1 methanol:water extraction for 96 h was used to extract formed dye from hair dyed with four different copper levels, and the absorbance signal correlated with copper levels in hair. The dye (Z)-5-amino-4- ((2-amino-1-(2-hydroxyethyl)-1H-pyrrol-3-yl)imino)-2-methylcyclohexa- 2,5-dien- 1-one was also synthesized and confi rmed to have the same 477-nm absorbance. A negative linear correlation of copper level with absorbance at 477 nm was measured with an R2 of 87% (Figure 6). The data confi rmed lower dye formation in hair pretreated with copper and explain the lighter color measured. A previous work has shown that copper taken up by hair from tap water accumulates in cuticle outer layers (6), and thus, it is proposed that more color is formed in the cuticle and outside hair when copper is present, Figure 3. Oxidation of primary intermediate to form diimine. Table I Metal Levels of Hair Tresses as Measured by ICP-OES (Standard Error in Parenthesis) Tress set Copper levels (μg/g) Calcium levels (μg/g) Magnesium levels (μg/g) 1 21 (1.8) 4,339 (55) 515 (7) 2 35 (0.8) 4,526 (36) 517 (3) 3 52 (1.7) 4,426 (78) 534 (8) 4 73 (2.0) 4,713 (54) 535 (7) JOURNAL OF COSMETIC SCIENCE 274