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

Figure 4. (A) L*, (B) a*, and (C) b* for three shades on copper-treated hair as a function of copper levels. REDOX METALS IN COLOR FORMATION IN A HAIR COLORANT 275