Hatao M, Murakami N, Sakamoto K, Ohnuma M, Matsushige C, Kakishima H, Ogawa T, Kojima H, Matsukawa K, Masuda K, Chiba K, Yoshizawa K, Kaneko T, Iwabuchi Y, Matsushima Y, Momma J, Ohno Y
Japan Cosmetic Industry Association (JCIA), Hatsumei Bldg, 9-14, Toranomon 2-chome, Minato-ku, TokyoJapan; Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 223Japan.
Toxicol In Vitro. 1999 Feb;13(1):125-37. doi: 10.1016/s0887-2333(98)00067-8.
Interlaboratory validation of the haemoglobin denaturation (HD) test on 38 cosmetic ingredients was conducted by five to eight participating laboratories. The HD test was evaluated as an alternative method to the Draize eye irritation test (Draize test) based on three indices of protein denaturation: the test substance concentration that induces 50% HD of the positive control (RDC(50)), a relative HD rate at 1% of the test substance (1%RDR) and a relative change in maximum absorption wavelength (1% lambdamax). The coefficients of variation associated with a positive HD test among the participating laboratories were within an acceptable range for practical application. The in vitro test results were in relatively good agreement with the Draize test. The correlation coefficient (r) between the in vivo maximal average Draize total score (MAS) and log (RDC(50)), 1%RDR and 1% lambdamax were -0.91, 0.67 and 0.79, respectively. The results revealed several limitations associated with the HD test: (1) the HD test cannot be applied to coloured test substances with a strong absorption, around 418nm; (2) water-insoluble test substances cannot be evaluated by RDC(50) or 1%RDR; (3) the HD test cannot be applied to strong acids that exceed the buffering capacity of a phosphate buffer solution; (4) the HD test cannot be used to determine the potential for eye irritation caused by factors other than protein denaturation, for example, polyoxyethylene octylphenylether (10 E.O.). Thus, the HD test alone is not appropriate for predicting eye irritation potential. Nevertheless, the good agreement between the HD test results and in vivo irritation scores as well as the ease of application suggest that this test may play an important role in a test system to determine eye irritation potential.
五至八个参与实验室对38种化妆品成分进行了血红蛋白变性（HD）试验的实验室间验证。基于蛋白质变性的三个指标，将HD试验评估为替代兔眼刺激试验（Draize试验）的方法：诱导阳性对照50%血红蛋白变性的受试物浓度（RDC(50)）、受试物1%时的相对血红蛋白变性率（1%RDR）以及最大吸收波长的相对变化（1%λmax）。参与实验室中HD试验呈阳性的变异系数在实际应用的可接受范围内。体外试验结果与Draize试验结果相对吻合。体内最大平均Draize总分（MAS）与log（RDC(50)）、1%RDR和1%λmax之间的相关系数（r）分别为-0.91、0.67和0.79。结果揭示了HD试验存在的几个局限性：（1）HD试验不适用于在418nm左右有强吸收的有色受试物；（2）水不溶性受试物不能通过RDC(50)或1%RDR进行评估；（3）HD试验不适用于超过磷酸盐缓冲溶液缓冲能力的强酸；（4）HD试验不能用于确定除蛋白质变性以外的因素（如聚氧乙烯辛基苯基醚（10 E.O.））引起的眼刺激可能性。因此，仅HD试验不适用于预测眼刺激可能性。然而，HD试验结果与体内刺激评分之间的良好吻合以及易于应用表明，该试验可能在确定眼刺激可能性的试验系统中发挥重要作用。
