WG８　Ｉｒｒｉｔａｔｉｏｎ　ａｎｄ　Ｓｅｎｓｉｔｉｚａｔｉｏｎ スウェーデンから、ISO 10993-10 Test for Irritation and Sensitizationの改訂要求が 提出され、それを受理するかどうかの投票が行われている。その結果次第で、 次回ヨーク会議（１９９７．４）で改訂作業が開始されるかもしれない。 日本は、反対票を投じた。改訂要求文書と日本の反対理由は下記のとおりである。 　　*********************************************************************　 　　　２月１９日付けで投票結果の報告があった。 　　　賛成　１６　反対　４（日本、英国、ドイツ、ポーランド） 　　　その結果、４月２３，２４日にＹｏｒｋで会議が持たれることになった。 　　　議長は、前議長のMike Liggett (UK)が再任される予定。 ********************************************************************* 「スウエーデンからの改訂提案書」 (Reasons for revision) The methods described in the standard are for many situations inadequate, especially for testing of mixtures of compounds: The Maximization sensitization test was designed for single chemicals - not mixtures. The Closed patch test sensitization test is sometimes used for formulated products, but the value of the Buehler test under these test conditions has not been documented in a scientific report and no comparative studies have been published. The Buehler test is claimed to be a screening test using a human model, i.e. Human repeat insult patch test (HRIPT). It can be suumed that an extract from a medical device is a chemical mixture. The recommended methods in present version of the standard might therefore not be optimal. The recommended methods for mixtures of chemical/fomulated products are the Open Epicutaneous Test (OET) or the Cumulative Contact Enhancement Test (CCET), both mimic the actual use situation. These and other alternative methods are presented in a monograph as ref.27 in the document. The OET is considered less sensitive than the CCET as it avoids the use of Freund's complete adjuvant (FCA). The in the document (pp3-5) recommended skin irritation test is not the most adequate test method available and uses animal experiments unnecessarily. The document lacks recommendations on the use of statistical methods for the evaluation of the results. (Suggested improvements) The procedure should be preceded by chemical analyses of the extract media (polar as well as non-polar solvents) in which the devices have been placed in order to demonstrate that a signal or mixture of chemicals are present in the extract. Appropriate analytical techniques should be selected for these analyses. If a single chemical is demonstrated the previously suggested methods (GPMT or Buehler test) are recommended. If more than one chemical is present in the extract the OET or the CCET, desribed above, are recommended. If the chemical analyses are negative, it is recommended not to carry out animal studies. it is a waste of resources. From an ethical point of view an animal study on a pure extract medium is highly questionable. A statistical analyses is highly recommended to complete the reactivity (numbers of reactions and their intensity) in actively induced animals and in control animals. However, if a reliable sensitization test according to the principles above has failed to demonstrate any allergic potential, it is suggested to carry out a skin irritation test in a human model. Thereby, the extrapolation problem (rabbit skin to man) can be avoided. By using modern, objective, non-invasive bioengineering techniques (laser doppler flowmetry, Minolta camera, evaporimetry, ultrasound etc) one can avoid the problems related to the reading of test reactions in rabbits, described by Weil & Scala (ref.17, page 29). ----------------------------------------------- 「日本の反対理由」 THE REASONS FOR OUR DISAGREEMENT for THE PROPOSAL OF REVISION OF ISO 10993-10 Japanese experts do not agree with "the reasons for revision" presented in the Swedish proposal. 1. About INADEQUACY OF USING MAXMIZATION TEST FOR TESTING MIXTURES OF COMPOUNDS: The proposal says "The Maximization test was DESIGNED for single chemicals - not mixtures." Even though it was correct, the "maximization test" has been USED to identify sensitization potencies of chemicals, chemical mixtures, and chemical formulations (such as cosmetics). We consider that its usefulness has been historically verified. In addition, please note the following: (a) In the test procedure of "maximization test", we often use the solution of the test chemical in an appropriate medium such as olive oil and dimethylsulfoxide (DMSO). Is not the solution a chemical mixture ? (b) There are many reports of tests of chemical mixtures by "maximization test" method, e.g. dyestuffs. It has been well known that Brilliant Lake Red R caused pigmented cosmetic dermatitis (PCD), and it contains a low level (530 ppm) of the impurity, Sudan I, that was finally clarified to be the cause of PCD with this dyestuff. Sato et al. (Contact Dermatitis, 1981: 7: 225-237 ) tested the dyestuff according to the modified maximization technique, that is called in Japan "Adjuvant and patch test method", and 10% (100,000 ppm) acetone solution gave positive reactions in 14/30 animals (MR=0.8). On the other hand, the strong sensitization potency of Sudan I (the minimum induction concentration = 10 ppm) was proven by the animal test. This paper shows the ADEQUACY of testing CHEMICAL MIXTURES by guinea pig test methods, such as maximization test, and the importance of the concentration of the causative chemical (Sudan I) in the test sample (Brilliant Lake Red R). This paper also reported that the test result of the dyestuff turned to be negative when tested after rinsing it with acetone. (c) ISO 10993-10 describes as follows: "6. Sensitization test ------ The maximization test is regarded as the most sensitive and is the preferred method, particularly with regard to the evaluation of extracts." Is the maximization test method adequate for evaluating the extracts ? The following papers show the adequacy when the extraction and test were appropriately done. (1) Kojima et al. (Contact Dermatitis, 1990: 23, 129-141) succeeded to sensitize guinea pigs with the acetone extract of the sweater, that caused severe allergic contact dermatitis among users. By patch testing of chromatographic fractions of the extract on the sensitized animal model thus prepared, they identified a new chemical (PCPHs) to be causative to the allergy; (2) In the same manner, Kaniwa et al. (Contact Dermatitis, 1992: 27: 166-173) succeeded to sensitize animals with the organic solvent (acetone/chloroform 1:1) extract of the rubber boots, that developed severe allergic contact dermatitis for the user. It was clarified that mercapto- benzothiazole (MBT) and its disulfide (MBTS) should be causative. 2. THE ADEQAUCY OF TEST depends upon the ADEQAUCY OF EXTRACTION AND APPLICATION OF THE EXTRACT TO THE TEST, not upon the TEST PROCEDURE itself. What is the adeqaucy of sensitization test in ISO 10993 series ? The test should serve to exclude the hazardous materials/devices from the market. Therefore, the test must not give false-negative results when applied to the devices that often cause the allergic dermatitis among the users. NOTE 15 of B.2.10 (Extraction media) of Part 10 says "Solvent extraction methods may be appropriate for skin sensitization." and B.4.5 describes "An alternative sample extraction procedure using a volatile solvent, followed by evaporation of the solvent and application of the residue to animals, may be undertaken for polymeric materials for sensitization testing." These are very important. Nakamura et al. (unpublished data) tried to sensitize animals with the extracts of the sweater and rubber boots described above (1.(c).(1) & (2)) using physiological saline and olive oil as recommended in B.2.10 as well as in ASTM. However, when tested the physiological saline extracts, no positive reactions were found; and when tested the olive oil extracts, positive reactions of the same grade were found in both of the test and control groups. Negative results by the application of the saline extracts were due to the lack of the causative chemicals (PCPHs, and MBT and MBTS) in the extracts. The false-positive reactions in control group are frequently found when applied vegetable oil extracts to the test. It is not appropriate to use vegetable oil as a solvent in challenge phase when it is used in the induction phase. In any test groups of these two experiments, any positive reactions were found when applied the acetone solutions of PCPHs and MBT (or MBTS) for challenge. These indicate that any animals were not sensitized with the causative chemicals of the sweater or the rubber boots. That means the tests were NOT ADEQUATE because of the INADEQUATE EXTRACTION, not because of the inadequacy of the animal test methodology. 3. INADEQUACY OF THE SUGGESTED IMPROVEMENTS In most cases of chemical analysis of the device materials, we detect multiple peaks in the chromatogram. Negative analytical results are rare. So, according to the suggestion, we have to usually proceed to the OET or the CCET. We still consider that the language of Clause 6 cited in 1.(c) is correct. Accordingly, we need no revision for the test method. 4. About including HUMAN MODEL We do not agree to include HUMAN MODEL for detecting the allergenic potentials of the materials, because (1) we cannot increase the concentration of the chemical for test for preventing unexpected hazards; (2) if the material contains a novel sensitizer that humans have never met, any positive reactions should not be expected because nobody was sensitized with such chemical, when the chemical concentration adopted for the test was below the minimum sensitization concentration; (3) to detect 5% significance of positiveness by the human patch test, more than 100 volunteers must be collected for each of test and control groups. For understanding our rationales, please refer ISO/TC194 N213 document delivered in Stockholm meeting, in 1996, and the Part 2 of the English translation of the Japanese Guidelines attached.