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1. 4 kay amp Calandra Ohno et al 1999 0 15 15 25 25 50 50 110 29 MAS 15 Mr E Ohno 2004 m 5 1 12. 5 No 28 No 77 2 40 60 2 1 3 2 1 40 60 2 91 2 2
3. Table 1 The effect of microplate sealing tape and wrap film in the LDM MTT assay Substances OD ae Without microplate sealing tape Negative control DW 0 306 100 and wrap film 20 triethanolamine 0 018 5 9 10 trietanolamine 0 059 19 3 5 triethanolamine 0 157 51 3 0 1 SDS 0 005 1 6 0 01 SDS 0 31 101 3 With microplate sealing tape Negative control DW 0 264 100 and wrap film 20 triethanolamine 0 015 5 7 10 trietanolamine 0 055 20 8 5 triethanolamine 0 155 58 7 0 1 SDS 0 012 4 5 0 01 SDS 0 348 131 8 48 Appendix 7 SDS Ze v SH ROT MATREX BEAIVIBOSE ROT 23540 SDS 1 1 PERLE XT ROT Z O
4. Ohno et al 1999 5 4 SOP 10 Table 3 List of the co operating organizations for the Japanese validaton Administrative organtzations Ministry of Health and Welfare National Institute of Health Sciences Div Pharmacol Div Toxicol and Div Genetics Mutagen Universities Yokohama City University Showa University Kit suppliers Oriental Yeast Lid Kurabo Industries Lid Invitro International Japan Ltd Toyobo Ltd Uthers RIKEN Gene Bank Japan Seigiken Rescarch Centre Co Ltd Japan Cosmetic Industry Corpor
5. Draize 110 80 Hagino et al 1991 GHS NI GHS NI MATREXM
6. Table 1 1 The Quality data of MATREX No Date Lot No Shape OD of untreated 50 of Pre shipment examination LDM SDS I0 12 o 1 jo 6 1t 19 oi 3 i2 31 Oi 1 o 1677 Acceptable 0200 000 Acceptable 7 19 0 0 1i 10 is 02 15 42 Tape 1 2 The Quality data of MATREX Date Lot No Shape OD 50 of Pre shipment examination SDS 01 03 022103 03 21 03 03 31 05 04 ji o 66 05 16 03 8 05 30 03 D3315 Acceptable 038 0 019 Accepabe 06 06 03 333 Acceptable 046 oos Accepabe 06 13 05 07 jn o 07 js 03 15 03 09 15 03 36 10 17 03 535533 Acceptable 039 9013 xcspsse I0 31 03 ii 703 53574 Accepable 0308 9017 Acepabe 2 12 o 43 1 3 The Quality data of MATREX Date Lot No Shape OD aia 50 of Pre shipment examination SDS wa di Acceptable erp pae ooa 6 04 04122 ______0352 oos 119 04 D4374 Acceptable BE 0 367 0 0 19 Acceptable i 04 D4401 Acceptable 0 311 0 016 Acceptable 121 04 D4409 Acceptable 0 382 0 019 Acceptable 122 04 D4416 ER lt
7. E Fig 7 Organization of the second and third validations Validation Management Committee Discussion and approval of plan validation program protocol SOP test chemicals in vivo test Publication of the Results Validation Expert Committee Planning validation Slee ol ame d per ipn Communication Scene Asset Group i Vivo Tes Cor Group Discuss evaluation Selection and management of test Planning of in vivo test chemicals Preparation of SOP Inspect contract laboratory m j Cosmetic Industry Association A Study Director B Study Director C Study Director Order in vive test Preparation of SOP Technology Contract Laboratry Management Data collection and preliminary analysis In vivo Test Preparation of P Fartopatng Experiment Experiment Experiment The figure is the same as that reported by Ohno et al 1999 The facilities of the study directors had the role of lead laboratory 12 GLP 1992 GLP
8. RARO 3 3 1 MATREX MATREX pH 6 8 7 7 Living dermal model LOM i5 LDM LDM LDM
9. 1999 Ohno 2004 MATREX Draize Ohuchi et al 1999 Globally belling of Chemicals GHS ND MAS15 MATREX GHS NI GHS NI Harmonized System of Classification and La Egi 2 Draize MATREX L lt Fi
10. Sn LDM 20 37 RAC 506 DMSO DLDM 6 LDM 5mL E
11. Ohuchi 1999 Appendix 4 Fig 1 Microscopic inspection of LDM M LDM Polycarbonate membrane The specimen of LDM was stained with hematoxylin and eosin The picture was taken by Toyobo Co Ltd on July 26 10 ned by nile blue RI K Fig 2 Fibroblast cell in LDM stai at MM Sail The picture is the same as that reported by Ishibashi 1996 5 3 LDM Fig 3 10mm 20mm Fig 4 216 Appendix 5 Fig 3 Cross section of MATREX Transwell Mesa region Assay plate Assay medium Polycarbonate membrane Fig 4 M
12. 39 GHS 34 No 1 No 34 2 cae 5 No 35 No 39 MATREXW Polyethylene glycol 400 3 4 19 Table 10 Results of interlaboratory reproducibility the LDM MTT assay Substance In vivo In vitro results classification Lab A Lab B Lab C Lab D Lab E Lab F Lab G 1 2 Ethylhexyl p dimethylamino N N N N N N N N benzoate 2 Isopropyl myristate N N N N N N N N 3 Isotonic sodium chloride N N N N sol
13. IC50 5 yo 1 BERD ARE v aV SERRE S 3 2 2 1 1 SDS ORERE 17 EE RRA AV 0 1 SDS FAVS 0 1 SDS 0 1 2 Eoo HG H3 188 0 7 7v E37 5 12 V 0 01 w v SDS 23 0 25 50
14. ZAN E MAS 5 Draize MAS15 5 Ohno 2004 0 5 5 25 25 50 50 110 88 Appendix 2 SIRC HeLa CHL
15. 1 2 AX5 10 A amp EE VERRE EZR EE ae REED 1 2 RATED 1 2 2 20 M a R Hee N B m m m E 3 Draize Draize
16. 2 1 1 5 5 3 20 2 2 2 1 1 99 1 1 1 1 BAEK 1 1 1 31 44 2 Table 12 The 77 substances Substance CAS Class Physical state MW 1 Ethyl 2 methyl acetoacetate 609 14 3 Esters Liquid 144 2 2 Ammonium nitrate 6484 52 2 Inorganic salts Solid 80 0 3 Butyl Dipropasol S
17. 1 2008 1 3 0 1mL 100mg 1 e 1 24 48 72 96 2 Draize 0 1mL
18. 2 Q Dodecylthio ethanol iso Octylthioglycolate MATREX 50 MTT 2 MTT Appendix 9 2 MATREX MATREX Diethylhexyl adipate Ethylhexyl palmitate Isocetyl stearate Isopropyl myristate Isopropyl palmitate Oleyl alcohol Safflower oil Sesame oil Sorbitan oleate Sorbitan sesquioleate Decyl oleate Ethylhexyl Stearate Castor seed oil 13 MTT LDM MTT Appendix 9 MTT
19. NI 1 GHS 77 F 2 P FE 1 1 4 E FE 1 m Phenylenediamine 2 n Dodecylthio ethanol H E 4 EDER IH iso Octylthioglycolate MTT Benzalkonium chloride Polyoxyethylene octylphenyle ther 10 E O Triton X 100 Isopropyl myristate Di 2 ethylhexyl sodium sulfosuccinate Dioctyl sodium sulfosuccinate 4 104 MA
20. 0 96 Ohno 1999 MATREXTY SIRC 0 81 Table 1 Correlation of the results between alternatives methods in the Japanese validation study HET CAM CAM TB RBC HET CAM 1 000 og CAM TB 0 679 1 000 RBC 0 653 0 743 1 000 SKIN M 0 479 0 811 0 953 0 628 0 733 0 936 CorePack 0 480 0 690 0 892 SIRC CVS 0 582 0 820 0 809 SIRC NRU 0 589 0 823 0814 HeLa MTT 0 580 0 831 0838 CHL CVS 0 545 0 765 0811 EYTEX 0 751 0313 0 542 The figure is the same as that reported by Ohno et al 1999 SKIN7 MATREX CornePack 1 000 0 931 0 846 0 838 0 821 0 872 0 841 0 188 1 000 0 913 0 814 0 840 0 820 0 389 1 000 0 773 0 768 0 812 0 798 0 202 SIRC SIRC CYS NRU 1 000 0 997 1 000 0 985 0 985 0 972 0 968 0 397 0 391 CHL MT
21. Table 2 1 Results of the MTT reduction by the 77 substances In vivo MTT No Substance classification In vitro results reduction 1 Ethyl 2 methyl acetoacetate 2 Ammonium nitrate 3 Butyl Dipropasol Solvent P P 4 3 Chloropropionitrile P P 5 Cyclopentanol P P 6 3 3 Dithiodipropionic acid P P a Hexyl cinnamic aldehyde 12 5 ir 8 N Lauroylsarcosine sodium salt 9 ak 10 2 Methyl I pentanol P P 11 Propasol Solvent P P 12 6 Methyl purine P P 13 2 6 Dichloro 5 fluoro beta oxo 3 pyridinepropanoate 14 Triton X 100 15 iso Octyl acrylate N 16 Tetra Aminopyrimidine sulfate N P 17 2 4 Difluoronitrobenzene N 6 18 n n Dimethylguanidine sulfate N P 19 2 n Dodecylthio ethanol N NE 20 iso Propyl bromide N P 21 Di iso butyl ketone N P 22 iso Octylthioglycolate N NE 23 2 4 Pentanediol N P 24 2 2 Dimethyl 3 pentanol N P 25 Potassium tetrafluoroborate N P 26 3 Methoxy 1 2 propanediol N P 27 Toluene N ae The color of the MTT medium changed to blue purple significantly and the MTT reduction was evaluated as positive The color of the MTT medium or the substance changed slightly The color of the MTT medium did not change and the MTT reduction
22. 5 E MATREX Y E E 50
23. 3 2 2 f 1 6 3 3 5 LDM LDM MTT EHAO 0 30 Sodium dodecyl sulfate SDS IC50 0 01 0 02 LDM 1 5mL 1 2mL
24. Appendix 15 7 3 2 6 3 2 JC QA 7 3 3 11 LDM LDM MTT 0 30 Positive Control Test Sodium dodecyl sulfate SDS IC50 0 01 0 02
25. 69 X 1 MATREX Y 6 3 0 01 SDS 0 1 SDS No 1 fT A Column 1 No 2 fT A Column 2 No 3 fT B Column 1 No 4 fT B Column 2 No 5 fT C Column 1 No 6 47 C Column 2 x105
26. Living dermal model LDM Bell et al MATREX 1 1991 Gay et al 1992 A 18 1996 24 MTT Haze LT FH v RI ERE RIL SIRC
27. 2 LDM 37 C 24 37 24 1 CPC 1 2mL 3 MTT 0 04N H n fi 1 Cetylpyridi MATREXT 37 24 1 Cetylpyridinium ch JL 1 6 Fig 2 MATREX H a cov ERR nium chloride C
28. HeLa CHL SIRC Ohno 1999 MATREX ik Appendix 2 5 MATREX SIRC Draize A 5 1 MATREX MATREX 2007 MATREX Hr SLE E 3 Bel
29. LDM 0 04N 0 3mL 2 0 2mL 96 570nm 1 EL HH 1mL 1 2mL BIEL 37 C C 3 0 04N 4 1 2 EFK 2 50 50 2
30. LDM 30 UH 5 1 2mL LDM 80 80mg LDM TEE 13 5 REII 37 24 5 0 1 w V SDS jv V5 Le SN 7 0 01 w v SDS 0 01 SDS
31. 1 LDM Table 9 TEN 50 P 50 4 1994 2 4 E 50 E 7 Lab E Lab F Polyethylene glycol 400 Lab E 1
32. GHS Global Harmonized System ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods IC50 Half Maximal 50 Inhibitory Concentration 50 OD Optical density OECD Organisation for Economic Co operation and Development MAS Maximal Average Draize Total Socre MTT 3 4 5 Dimethylthiazol 2 yl 2 5 diphenyltetrazoliu m Bromide QA Quality Assurance 85 Appendix 1 Draize 1 Draize Draize 1981 OECD 1987 2002 OECD 2002 Draize 2008 2008
33. Results of the first measurement Results oF the second MTTassay In No Substance ORTS E T BENE inter rence vitro iro Evaluation Viablity 0 Positive P or Viablity 96 Positive P or Corrected classifcaiton Negative N Negative N viability Iesults 19 2 n Dodecylthio ethanol 81 2 N 118 9 N N False positive Intensity 0 1 44 6 22 iso Octylthioglycolate 171 3 N 141 3 N N Not evaluated Intensity 56 3 NE 33 Butylene glycol 71 8 N N N N True negative 39 Diethylhexyl adipate 107 1 N N N N True negative 42 Ethylhexyl palmitate 77 2 N N N N True negative 44 Isocetyl stearate 00 9 N N N N True negative 45 Isopropyl Myristate 98 4 N N N N True negative 46 Isopropyl Palmitate 02 8 N N N N True negative 49 Oleyl alcohol 89 0 N N N N True negative 53 Phytantriol 58 1 N 55 2 N N N False negative 56 Safflower oil 104 9 N N N N True negative 57 Sesame ol 01 3 N N N N True negative 61 Sorbitan oleate 63 3 N 71 8 N N N N True negative 62 Sorbitan sesquioleate 58 9 N 85 8 N N N N True negative 63 Squalane 69 2 N N N N True negative 67 Triethylene glycol 77 0 N N N N True negative 69 Acetyl tributyl citrate 27 3 N N N N True negative 71 Decyl oleate 06 4 N N N N True negative 72 Ethylhexyl stearate 91 0 N N N N True negative 73 Glyceryl stearate 01 3 N N N N True negative 74 PEG 2 stearate 85 3 N N N N True negative 75 Castor seed oil 99 9 N N N N True negative
34. 1 MATREX GHS NI LDM 50 50 NT NI MATREX Y GHS NI 10 Bell E Ivarsson B and Merrill C 1979 Production of a tissue like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential zn vitro Proc Natl Acad Sci USA 76 1274 1278 Bell E Parenteau N Gay R Nolte C
35. NI GHS 28 NI 25 Reference Substance List 2006 2B NI 27 MATREX 100 50 MATREX MATREX GHS NI 7 2 GLP GLP 7 3 7 3 1
36. M MAS 15 3 Draize E Draize Draize 1959 Draize 1996 IRO GHS 1 2A 2B NI Draize Table 1 GHS
37. 0 01 SDS N SON 0 01 SDS 0 25 50 H 0 333mg mL MTT 1 5mL 1 2mL IC50 H IC50 50 IC50 50 50 MAS15 1C50 1 2
38. 6 sr z 0 25 50 0 01 SDS 0 25 11 j lt N e Ie TH ar H 50 7 MTT LDM CaS 0 1 SDS 0 1 LDM 3mL 41 15 LDM LDM MTT 0 333 06 8mm
39. 68 O WKL216 TSQ456 Wako Alfa aesar TCK0946 9 8 9 eee mew PT 6 9 Wal oo E 1 6 5 VER PO 7 I s Ws x pem zoe se 59 532 02 5 963 owe MP Biomedicals ERATE _ ieee Wako TEA ee peers SADT 67 112 27 6 3 6 1 8 Wako WKG5787 68 122 20 3 PBNI131 69 77 90 7 O VEA EV w AF TIAA 2 Wt rr serm evo OSS Wa Sigma Aldrich Sigma Aldrich Corp TCI Wako
40. 39 18 3 3 3 3 2 1 PABA 1 1 20 15 4 18 10 fi a BR 35 1 3 0 1 1 3 3 2 12 1 24 18 3 2 2
41. 6 3 2 LDM MTT XT Ze TE SAI LDM 1 MATREX MATREX pH 6 8 7 7 3 LDM EO LDM pH 0 30 SE TU7 Sodium dodecyl sulfate SDS IC50 0 01 0 02 C RE TD
42. Table 1 Results of repeatability on the LDM MTT assay 50 j Lab F 1 2 3 1 E 2 2 E 2 Substance In vivo 1C50 96 and in vitro classification classification Lab A Lab B Lab C Lab D Lab E Lab F Lab G 2 Ethylhexyl N 5100 5100 5100 5100 5100 5100 5100 p dimethylamino N N N N N N N Benzoate 5100 5100 5100 5100 5100 5100 5100 N N N N N N N Isopropyl N 5100 5100 5100 5100 5100 5100 5100 N N N N N N N Myristate 5100 5100 5100 5100 5100 5100 5100 N N N N N N N Silicic anhydride N 5100 5100 5100 550 gt 50 gt 100 gt 100 N N N NC NC N N 5100 5100 5100 5100 5100 5100 5100 N N N N N N N Polyethylene glycol 400 N 5100 5100 Around 75 Around 75 75 gt 100 75 100 N N P P P N P 5100 5100 85 76 5100 67 82 N N P P N P
43. 0 3 Sodium dodecyl sulfate SDS IC50 0 01 0 02 LDM 20 37 30 C OF 3 2 MCO 17AIC CN 25A CCV1300E Benchmark Plus 3 3 8mm
44. Kay amp Calandra 1962 Guillot 1982 DeSousa 1984 ET 3 Kay amp Calandra 0 0 5 0 5 2 5 2 5 15 15 25 25 50 50 80 80 100 100 110 37 Ohno 1999 Kay amp Calandra Kay amp Calandra 8 Draize MAS15 f LLTD
45. 3 2 2 Table 8 Draize eye test results as is in the Japanese validation study including the classification predicted from the result of 10 concentration GHS NI 9 34 34 14 3 2 PABA 1 FAN 1 1 GHS classification based GHS classification by Substance MAS on the data of Japanese ICCVAM Recommended ed classification validation study Reference Substance List 1 2 Ethylhexyl p dimethylamino benzoate 0 0 2 Isopropyl myristate 0 0 3 Isotonic sodium chloride solution 0 0 4 Silicic anhydride 2 7 5 Polyethylene glycol 400 4 0 6 Glycerin 4 7 7 Polyoxyethylene sorbitan monooleate 20E O 47 8 Triethanolamine 8 0 9 Methyl p hydroxybenzoate 8 7 10 Sucrose fatty acid ester 28
46. GHS NI 29 Table 15 The eye irritancy of test samples predicted by LDM MTT assay In vitro Classification by SIRC CVS assay using triethanolamine as a reference substance for non irritancy Positive Negative In vivo Positive 56 1 Classification by 1 2Aor 2B Sucrose fatty acid ester Phytantriol Benzyl alcohol GHS Ethanol Acid red 92 Calcium thioglycolateSodium salicylate Distearyldimethylammonium chloride Lactic acid Sodium dodecyl sulfate Diisopropanolamine Monoethanolamine Glycolic acid Sodium hydrogenated tallow L glutamate Chlorhexidine gluconate 20 solution Butanol Potassium laurate Polyoxyethylene octylphenylether 10 E O Di 2 ethylhexyl sodium sulfosuccinate Acetic acid Cetyltrimethylammonium bromide Benzalkonium chloride Stearyltrimethylammonium chloride Cetylpyridinium chloride Domiphen bromide Ethyl 2 methyl acetoacetate Ammonium nitrate Butyl Dipropasol Solvent 3 Chloropropionitrile Cyclopentanol 3 3 Dithiodipropionic acid Hexyl cinnamic aldehyde 12 5 N Lauro
47. MATREX Y 30 MTT E T N N 100 T The viability of LDM treated with the substance LDM N The viability of LDM of negative control LDM 30 MAT OT EEK T T CT CN REX M 1 CPC 0 1 MTT FE T The viability of LDM treated with the substance 52 H LDM
48. 28 Benzalkonium chloride Polyoxyethylene octylphenylether 10 E O Isopropyl myristate Di 2 ethylhexyl sodium sulfosuccinate MATREX Y Appendix 13 104 81 Phytantriol 1 3 2 Bromo 2 Nitropropane 1 3 Diol Hexylene glycol Phytantoriol 6 Glycerin Polyethylene glycol 400 2 4 Pentanediol 3 Methoxy 1 2 propanediol Butylene glycol Triethylene glycol 9 5 Polyethylene glycol 400 2 4 Pentanediol 3 Methoxy 1 2 propanediol Phytantriol Polyethylene glycol 400 Polyoxyethylene sorbitan monooleate 20E O Triethanolamine Methyl p hydroxybenzoate iso Octyl acrylate tetra Aminopyrimidine sulfate 2 4 Difluoronitrobenzene n n Dimethylguanidine sulfate 2 n Dodecy
49. 50 KATA 50 j Lab F 1 8 B 2 3 1 2 2 E 2 R 5 50 N 50A EDGARA 7 Table 9 Results of repeatability on the LDM MTT assay Substance In vitro results ication Lab A Lab B Lab C TbD Lab E Lab F Lab G 2 Ethylhexyl N N N N N N N N p dimethylamino Benzoate N N N N N N N Isopropyl N N N N N N N N Myristate N N N N N N N Silicic anhydride N N N N NC NC N N N N N N N N N Polyethylene glycol 400 N N N ip N N N P P Glycerin N N N N N N N N N N N N N N N Polyoxyethylene N P P sorbitan monooleate 20 P P P P P P P Methyl N
50. MTT 30 MTT 30 50 50 TEs Fig 1 Layout of 6 well microplate at the test for detecting substances that interfere with MTT endpoint Negative control Sample l the LDM killed by 1 CPC non treated control or solvent Negative control 2 Sample2 the LDM killed by 1 CPC non treated control or solvent Control Sample3 the LDM killed the LDM killed by 1 CPC by 1 CPC Well No 1 Row A Column 1 No 2 Row B Column 1 No 3 Row C Column 1 No 4 Row A Column 2 No 5 Row B Column 2 No 6 Row C Column 2 53 Fig 2 Killing of LDM by using 1 CPC me T LDM after 24 hr application of the AE substance 1 Transwell 4 1 Treatment of 1 CPC for 8 d min Washing with DW MTT as
51. 0 01 SDS 0 25 50 0 1 SDS 0 1 4 5 2 50 50 No 1 No 27 2 2 3 2 No 28 No 77 40 60 1 1 40 60
52. RS 6330 96 200 u L 1000 u L 5mL GILSON 4 MICROMAN GILSON CP250 64 9793R G Sodium dodecyl sulfate SDS Dimethyl sulfoxide DMSO Ethylene glycol Liquid paraffin 3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide Isopropanol Hydrochloric acid 3 5 MTT 3 5 1 0 333mg mL MTT 0 333mg mL 3 5 2 0 04N HCl Isopropanol 12N HCI Isopropanol 0 04N Jp 3 6 water 50 v v DMSO Ethylene glycol Liquid paraffin 2 A BEI Jp 3 7 XTR WDA 3 7 1 0 1 w v SDS 3 7 2 PE xf HALA
53. 2 3 2 1C50 6 4 j IC50 39 Table 6 39 19 Jr 2 2
54. 2A 2B 16 11 2 1 2 ICCVAM Polyoxyethylene octylphenylether 10 E O ICCVAM GHS 1 10 1or2A Sodium dodecyl sulfate ICCVAM E 10 6 3 4 1or2A 1or2 15 25 2 Jv
55. 2 n Dodecylthio ethanol iso Octylthioglycolate MTT MTT Appendix 9 11 2 NI No 28 No 77 50 2008 LDM MTT V 1 MATREXT 123 E 71 8 CHV j 2 a ay JSAAE 20085 Phytantriol 4 21 E No 61 Sorbitan ol 3 50 RE
56. LDM 0 04N HCl Isopropanol 0 3mL 2 0 2mL 96 570nm 0 04N HCLIsopropanol 0 2mL 1 2 Bxcel 2 4 4 0 25 50
57. Table 2 GHS pH pH 2 11 5 pH 1 Tv T Table 1 Grading of ocular lesions Cornea Opacity degree of density readings should be taken from most dense area No ulceration or opacity Scattered or diffuse areas of opacity other than slight dulling of normal lustre details of iris clearly visible Easily discernible translucent area details of iris slightly obscured Nacrous area no details of iris visible size of pupil barely discernible Opaque cornea iris not discernible through the opacity Maximum possible 4 The area of corneal opacity should be noted Iris Mar
58. 2008 996 RUE 22 6 218 223 58 59 21 in vivo Appendix 14 1991 OECD j MATREX 22 6 211 217 j A 2008 SIRC 3 11
59. E 2 it 4 i X MTT 0 30 Positive Control Test Sodium dodecyl sulfate SDS IC50 0 01 0 02 2008 21 2 7 4 Table 12 oe ICCVAM Recommended Reference Substance List NI 2B 27 WA No 1 27 amp Hv vis 50 No 28 77 77 15 13 10 7 100 in vivo
60. 3 0 01 SDS 3 4 4 1 LDM ux 4 2 pore HERE E 0 01 w y SDS 30 1 2mL LDM Fig 1 3 80u L LDM 80mg jv V5 Jr AUC H 6 LDM Fr Pas REI 135 E Fe 2
61. 2 2 2 1 PABA 1 1 1 1 13 Table 6 List of the test substances and their characteristics Substance CAS Class Physical state MW lsotonic sodium chloride solution 7647 14 5 Inorganics Solution 58 4 Polyoxyethylene hydrogenated castor oil 60 E O 61788 85 0 Surfactants Solid Polyoxyethylene sorbitan monolaurate 20 E O 9005 64 5 Surfactants Liquid 346 5 Polyethyleneglycol monolaurate 10 E O 9004 81 3 Surfactants Liquid Sodium N lauryl sarcosinate 30 solution 137 16 6 Surfactants Solution 311 4 Sodium hydrogenated tallow L glutamate 68187 34 8 Surfactants Solid Sodium dodecyl sulfate 151 21 3 Surfactants Solid 288 4 Sodium polyoxyethylene laurylether sulfate 2 E O 3088 31 1 Surfactants Solution 274 4 27 solution Polyoxyethylene octylphenylether 10 E O 9002 93 1 Surfactants Liqui 324 4 Benzalkonium chloride 8001 54 5 Surfactants Soli 283 9 Sucrose fatty scid ester Surfactants Soli Glycerin 56 81 5 Polyols Liqui 92 1 Acid red 92 18472 87 2 Color additives Soli
62. ROVE eate O 3 E E Appendix 12 49 58 1 55 2 CY 50 MATREX GHS NI Table14 Results of the 77 substances Phytantriol 4 FH Results of the first measurement Results of the second In r measurement In vivo Substance vitro Evaluation Viablity 9 Positive Viablity Positive results classification Negative N Negative N 1 Ethyl 2 methyl acetoacetate 66 32 True positive 2 Ammonium nitrate 43 19 True positive 3 Butyl Dipropasol Solvent 13 1 24 P P P True positive 4 3 Chloropropionitrile 72 21 True 5 Cyclopentanol 13 1 12 P P P True 6 3 3 Dithiodipropionic acid 28 20 True 7 Hexyl cinnamic aldehyde 12 5 44 0 35 2 P P P True positive 8 N Lauroylsarcosine sodium salt 35 40 True 9 17 1 57 True i positive 10 2 Methyl 1 pentanol 62 16 P P P True positive 11 Propasol Solvent P 18 2 21 True
63. Substance as 15 di in MAS GHS iiid state classification 2 Ethylhexyl p dimethylamino benzonate Liquid 0 0 NI N Isopropyl myristate Liquid 0 0 NI N Isotonic sodium chloride solution Liquid 0 0 NI N Silicic anhydride Powder 2 4 NI N Polyethylene glycol 400 Liquid 4 0 NI N Glycerin Liquid 4 7 NI N Polyoxyethylene sorbitan monooleate 20 Liquid 4 7 NI N Triethanolamine Liquid 8 0 NI N Methyl p hydroxybenzoate Powder 8 7 NI N Sucrose fatty acid ester Powder 28 3 lor2A P Benzyl alcohol Liquid 31 0 lor2A P Ethanol Liquid 32 7 lor2A P Acid red 92 Powder 71 0 lor2A P Calcium thioglycolate Powder 79 7 1 P m Phenylenediamine Powder 80 7 lor2A P Sodium salicylate Powder 83 7 lor2A P Distearyldimethylammonium chloride Powder 96 3 1 P Lactic acid Liquid 102 7 1 P MAS Maximal average score of the Draize eye test GHS category 1 Severe or corrosive irritant 2A Irritant 2B Irritant NI Non irritant lor2A The Draize eye test results couldn t discriminate between 1 and 2A for no observation data on day 21 The observation was performed to day 14 P Positive N Negative 106 Draize GHS Table 8 10 1
64. 2 2 3 2 No 1 No 27 No 28 No 77 2008 21 50 40 60 66 Hagino S Okazaki Y Kitagaki M and Itagaki 2010 Further verification of an in vitro tier system for the identification of cosmetic ingredients that are not ocular irritants Alternatives to Laboratory Animals
65. oos Acceptable oos Acceptable Pas 125 863000 060 oms Aecpabe Pas 121208 863900 0378 oor Acceptable us 1 509 865500 oms ooms Acceptable Pais 1 9 09 865600 039 90 Acceptable 46 Fig 1 The distribution of OD of untreated control in the LDM MTT assay Frequency 3 0 32 034 036 038 04 042 044 046 048 05 052 0 54 0 56 OD of untreated control Fig 2 The distribution of IC50 of positive control in the LDM MTT assay Frequency 0 0 012 0012 0013 0 014 0015 0016 0017 0018 0 019 0 02 1C50 of SDS 47 Appendix 6 MATREX 24 DW 0 01 SDS 1
66. 29911 27 1 1 1 2 2 3 5 96 41 3 6 1119 62 6 3 3 11 12 3 105 30 6 2 1 2004 03 7 6 1H 96568 04 6 3 2 6 5 3 2 5 29590 42 9 16 5392 28 9 2 4 5 6 18 598 65 2 NN 1462 55 1 2 1569 01 3 1 2 3 9002 93 1 10 E O 7 446 35 5 2 4 75 26 3 108 83 8 2 6 4 625 69 4 2 4 FI N N VD 2 1 1 1 1C 1C rich
67. 2010 10 H 18 H MATREXTY 1 3 Draize Draize MATREX 5 MATREX 6 7 MATREX 2 8 MATREX 9 10 11 1 2008 1990 f 25
68. 5 IC50 50 7238 MATREX M 50 Bl 6 6 1 7 MATREX 2 2 KEKI R IC50 100 50 IC50 aa 1
69. Phytantriol Phyta ntriol 50 4 Table 1 1 E Table 1 Table 1 The comparison between the additional data and the previous data reported at the 21th annual meeting of the JSAAE No Substance in JSAAE data classification data 28 2 Bromo 2 Nitropropane 1 3 Diol P P P 29 Benzalkonium chloride P P P 30 Benzophenone 1 P P P 31 Benzophenone 2 P P P 32 Butoxyethanol P P P 33 Butylene glycol N N N 34 Cetrimonium chloride P P P 35 Cetyl alcohol N P P 36 Chlorhexidine digluconate 20 Solution P 37 Chlorophene P P P 38 Chloroxylenol P P P 39 Diethylhexyl adipate N N N 40 Diisopropyl adipate N P P 41 Dioctyl sodium sulfosuccinate P P P 42 Ethylhexyl palmitate N N N 43 Hexylene glycol P P P 44 Isocetyl stearate N N N 45 Isopropyl Myristate N N N 46 Isopropyl Palmitate N N N 47 Lauramide DEA P P P 48 Methoxyisopropyl acetate P P P 49 Oleyl alcohol N N N 50 40 stearate N P P 51 Phenethyl alcohol P P P 52 Phenoxyethanol P P P 53 Phytantriol P N P 54 Propy
70. MTT Monoethanolamine 4 4 MATREX MTT Table 1 Results of the MTT reduction by 34 substances used in the Japanese validation study Substance In vivo MATRES MTT Classifica test reduction tion results 1 2 Ethylhexyl p dimethylamino benzoate N N 2 Isopropyl myristate N N an 3 Isotonic sodium chloride solution N N 4 Silicic anhydride N N 5 Polyethylene glycol 400 N 6 Glycerin N N 7 Polyoxyethylene sorbitan monooleate 20E O N P 8 Triethanolamine N P s 9 Methyl p hydroxybenzoate N P 10 Sucrose fatty acid ester P P 11 Benzyl alcohol P P gl 12 Ethanol P P 13 Acid red 92 P P Red 14 Calcium thioglycolate P P 15 m Phenylenediamine P P 16 Sodium salicylate P P 17 Distearyldimethylammonium chloride P P 18 Lactic acid P P 19 Sodium dodecyl sulfate P P 20 Diisopropanolamine P P 21 Monoethanolamine P P 22 Glycolic acid P P 23 Sodium hydrogenated tallow L glutamate P P NT 24 Chlorhexidine gluconate 2
71. 27 S2 2008 49 Oleyl alcohol JACT 4 5 1 29 1985 50 PEG 40 stearate JACT 2 7 17 60 1983 51 Phenethyl alcohol JACT 9 2 165 83 1990 52 Phenoxyethanol JACT 9 2 259 77 1990 53 Phytantriol IJT 26 Suppl 1 107 117 2007 54 Propylene carbonate JACT 6 1 23 51 1987 55 Resorcinol JACT 5 3 167 203 1986 56 Safflower oil JACT 4 5 171 97 1985 57 Sesame oil JACT 12 3 261 77 1993 58 Sodium dehydroacetate JACT 4 3 123 159 1985 59 Sodium naphthalenesulfonate IJT 22 Suppl 2 37 44 2003 60 Sodium stearate JACT 1 2 143 77 1982 61 Sorbitan oleate JACT 4 3 65 121 1985 62 Sorbitan sesquioleate JACT 4 3 65 121 1985 63 Squalane JACT 1 2 37 56 1982 64 Stearalkonium chloride JACT 1 2 57 69 1982 65 TEA Lauryl sulfate 4096 Solution JACT 1 4 143 67 1982 66 Triacetin 22 S2 1 10 2003 67 Triethylene glycol 25 5 121 138 2006 68 Triisopropanolamine JACT 6 1 53 76 1987 69 Acetyl tributyl citrate IJT 21 S2 1 17 2002 70 Cetyl palmitate JACT 1 2 13 35 1982 71 Decyl oleate JACT 1 2 85 95 1982 72 Ethylhexyl stearate JACT 4 5 107 46 1985 73 Glyceryl stearate JACT 1 4 169 192 1982 74 PEG 2 stearate JACT 2 7 17 60 1983 75 Castor seed oil JACT 7 6 721 739 1988 76 Stearyl alcohol JACT 4 5 1 29 1985 77 Zinc stearate JACT 1 2 143 77 1982 IJT International Journal of Toxicology JACT Journal of the American College of T
72. 184 2 29 OS Si660O0 0018 Acceptable 185 3 7 208 si7400 _ 048 0017 Acceptable 16 3 14 08 817900 0014 Acceptable 187 3 21 208 821000 0421 0019 Acceptable 18 4 4 208 822200 046 0018 Acceptable 4 os 823000 042 0017 Acceptable 190 4 18 08 825800 047 000 Acceptable Fin 5 9 08 826500 0424 0017 Acceptable 192 5 30 08 830500 042 0015 Acceptable 13 6 6 208 831300 0387 0018 Acceptable 14 6 13 08 832200 0361 0019 Acceptable 195 6 20 835500 0 0320 0016 Acceptable 16 6 27 08 836400 0334 0015 Acceptable 197 7 18 08 840500 _ 0476 001 Accepable 18 7 25 08 841200 034 0018 Acceptable 19 8 1 os s lo0 047 0015 Acceptable 200 8 8 208 842700 045 0014 Acceptable of os oos Aceepeple ______ 202 08 845800 0 497 0 015 Acceptable Fas 838 oo 204 08 850800 0 501 0 015 Acceptable 205 10 08 851500 0 418 0 016 Acceptable 10 i 08 85200 Accepable 207 10 17 08 855500 0 430 0 017 Acceptable 208 10 24 08 856100 0 438 0 017 Acceptable 7 857700 048 907 xspsge na osf 858500 oos Acceptable 21 os
73. Appendix 4 Ohuchi MATREXW LDM Fig 1 Ohuchi 5 1999 Fig 1 Histological section of LDM The picture is the same as reported by Ohuchi et al 1999 41 Appendix 5 MATREXM Table 1 1 1 5 LDM OD Sodium dodecyl sulfate SLS IC50 10mm 20mm OD 0 30 SDS 1050 0 10 0 206 216 LDM OD 0 385 0 065 SLS IC50 0 016 0 002 Fig 1 Fig 2 216 LDM OD SLS IC50 HAE CARLIE
74. Draize MAS MAS 110 80 10 20 1996 E 36 2 Draize I A lt lt 1 4 1 4 1 2 1 2 A 3 4 3 4 A AXBX5 80 A 0 JER 1 1
75. E 0 01 SDS 0 1 SDS OD QCS0 MATREX Y Table 1 50 C50 N 50 P 50 7 Lab E Lab F Polyethylene glycol 400 50 1 4 E 50 Lab E 1 50
76. ation Shiseido Safety amp Analytical Res Center POLA Corp Kanebo Lid KOSE Corp Lion Cuip KAO SUNS TAR Inc OPPEN Cosmetic Co Ltd NOEVIR Co Ltd Kaminomoto Co Ltd Procter amp Gamble Far East Inc Nippon Cosmetic Co Ltd Yakult Central Institute for Microbiological Res Ajinomoto Co Inc Cow Brand Soap Kyoshinsha Co Ltd Hoyu Co Ltd CLUB COSMETICS Co Lid Nippon Shikizai Inc The table is the same as that reported by Ohno et al 1999 MATREX 3 7 P amp G 4 Table 4 List of the participation of organization First valication Kao Toyobo Lion Second validation Kao Toyobo Lion Noevir P amp G Menard Third validation Kao Toyobo Lion Noevir 10 6 3 MATREX 6 3 1
77. 00619PC ricn ricn ricn ricn ricn ricn ricn ricn ricn 09223 06127 8 1 175041 0 LB 1 049 1156 1 1 09620MU 118K0160 10428CH 15022HH MKAA4323 uka Sigma Aldric Sigma A Aldrich Sigma Aldrich Aldrich T ricn H 833971017 H 833977018 2 28 623 39 2 3 1 2 52 51 7 2 2 1 3 S 5 SSS 08216PE ETT ic i ic i ic i ic i ic ic ic ic ic ic Aldr Aldr Aldr Aldr S26421 416 Aldr Aldr Aldr Aldr Aldr h h h h h h h h h h Or mt RIAN 20 41 42 43 44 42 29806 73 3 43 107 41 5 44 25339 09 7 18472 51 0 111 76 2 2 ALP5483 Eo IE 2 2 4
78. 12 6 Methyl purine 268 38 P P P True positive 13 2 6 Dichloro 5 fluoro beta P P P True RE 6 6 6 9 Nh 3 positive 14 Triton X 100 38 12 True positive 15 iso Octyl acrylate 41 04 N Fal se positive 16 Tetra Aminopyrimidine sulfate 54 37 N False positive 24 Di ronitr als 17 2 4 Difluoronitrobenzene 10 7 10 P P N Fal se positive 18 n n Dimethylguanidine sulfate 94 13 P P N Fal se positive 19 2 n Dodecylthio ethanol 81 2 N 118 9 N NE N Could not be evaluated 20 iso Propyl bromide 3 8 6 0 P P N False positive 21 Di iso butyl ketone 2 6 32 P P N False positive 22 iso Octylthioglycolate 171 3 N 141 3 N NE N Could not be evaluated 2 Pentanedi als 23 2 4 Pentanediol 31 26 P P N Fal se positive 24 22 Dimethyl 3 pentanol 12 26 P P N False 25 Potassium tetrafluoroborate 22 46 N Fal Se positive 26 3 Methoxy 1 2 propanediol 10 1 13 0 P P N False 26 positive 27 Toluene 47 1 3 False positive 27 continued Results ofthe first measurement Results ihe second In measurement In vivo Substance Viablity Positive P or Viablity Positive niro classificai
79. ETE LDM OU 5 A 2 TI 3mL 15 4 65 37 C 24 oe BEKT Fy CASHES IPRAL SOIC LDM Distilled 1 1 1 MAREE CH VS 5mL F LDM Uv x VESOqZuyvw gt 4 3 MTT 0 333mg mL MTT 1 2mL 37 3 LDM 8mm
80. H 16CPC LDM CT The viability of LDM treated with 1 CPC following the substance application 16CPC CN The viability of LDM treated with 1 CPC following no substance application 1 CPC MTT 1 CPC 0 1 0 25 16CPC 0 1 MTT MATREX
81. Kemp P Bilbo P Ekstein B amp Johnson E 1991 The living skin equivalent Its manufacture its organotypic properties and its responses to irritants Toxicology in Vitro 5 591 596 DeSousa D J Rouse A A and Smolon W J 1984 Statistical consequences of reducing the number of rabbits utilized in eye irritation testing data on 67 petrochemicals Toxicology and Applied Pharmacology 76 2 234 42 Draize J H 1959 Dermal toxicity In Appraisal of the Safety of Chemicals in Food Drugs and Cosmetics Vol 46 The Association of Food and Drug Officials of the United States Austin TX ECETOC 1998 Technical report No 48 2 Eye irritaton Reference chemicals data bank Second Edition Gay R J Swiderek M Nelson D amp Stephens T J 1992 The living dermal equivalent as an in vitro model for predicting ocular irritation Journal of Toxicology Cutaneous amp Ocular Toxicology 11 47 68 33 Guillot J P Gonnet J F Clement C Caillard L and Truhaut R 1982 Evaluation of the ocular irritation potential of 56 compounds Food and Chemical Toxicology 20 5 573 82 Hagino S Itagaki Kato S Kobayashi T and Tanaka M 1991 Quantitative evaluation to predict eye irritancy of chemicals modification of chorioallantoic membrane test by using trypan blue Toxicology in Vitro 5 301 304 Hagino S Okazaki Y Kitagaki M and Itagaki H 2010 Further verification
82. 1991 4 MATREX 1 1980 FRZ Draize 1 LD50 oe Co P2 26 31 31 33 HOOK ORE E 6 18 29 MATREXWY 1999 4 1 E HAERES
83. M Inoue K Okumura H Arashima M Kurishita A Kinoshita S Tani N Kojima H Nakamura T Suzuki K Ishibashi T Hori H Takahashi H Nishikawa T Kitano Y amp Ohno Y 1999 Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients 6 Evaluation of MATREX Toxicology in Vitro 13 153 162 SIDS http www chem unep ch irptc sids oecdsids sidspub html third revised edition of the GHS published in July 2009 http www unece org trans danger publi ghs ghs rev03 03files e html 1996 MATREX 22 6 234 237 2008 44 9 863 868 34 1996 aE MATREX 1994 XH 1996 1999 1 7 21 26 2008
84. 0 359 0 019 Acceptable 4 gt 3 D4422 0 384 0 018 Acceptable 124 8 04 D4430 Acceptable 0 305 0 019 Acceptable 125 8 3 04 D4455 a 0 373 0 019 Acceptable I 10 6 44 Tape 1 4 The Quality data of MATREX Date Lot No Shape OD ES 50 of Pre shipment examination SDS Ex a 301 Acceptable 166 gt 07 746800 Acceptable BE 0 402 0 1016 Acceptable 168 07 752300 Acceptable 0 451 0 017 Acceptable 169 10 2 07 752900 S lt 0 461 0 015 Acceptable 10 07 171 10 07 756300 Acceptable 0 344 0 016 Acceptable 172 11 9 07 757300 Acceptable 0 316 0 014 Acceptable 11 16 707 760400 0 338 0 014 Acceptable 11 23 07 761200 Acceptable 0 342 0 014 Acceptable 11 30 07 761900 0 333 0 017 Acceptable 12 7 07 762900 0 398 0 015 Acceptable 12 14 07 763800 0 325 0 013 Acceptable 45 Tape 1 5 The Quality data of MATREX Date Lot No Shape OD 50 of Pre shipment examination SDS us 1 7 os 765900 0337 0 016 Accepable 179 1 11 08 766000 040 0015 Acceptable 2 1 208 81000 0428 0015 Acceptable 181 2 8 08 8II900 _ 0530 0019 Acceptable 182 2 15 O8 sso 0432 0019 Accepable lis3 2 22 08 sissoof oos Acceptable
85. 1 or 1 P 56 Safflower oil NI N 57 Sesame oil NI N 58 Sodium dehydroacetate NI N 59 Sodium naphthalenesulfonate 2A or 1 P 60 Sodium stearate NI N 61 Sorbitan oleate NI N 62 Sorbitan sesquioleate NI N 63 Squalane NI N 64 Stearalkonium chloride 2A or 1 P 65 TEA Lauryl sulfate 2A or 1 2A 1 66 Triacetin NI NP NP N 67 Triethylene glycol NI NT N 68 Triisopropanolamine 2B 2A or 69 Acetyl tributyl citrate NI N 70 Cetyl palmitate NI N 71 Decyl oleate NI N 72 Ethylhexyl stearate NI NT N 73 Glyceryl stearate NI N 74 PEG 2 stearate NI N 75 Castor seed oil NI N 76 Stearyl alcohol NI NE NP N TI Zinc stearate NI NT N Tested concentration is shown in parenthesis It was classified on the basis of the data from the IUCLID It was classified on the basis of the data from SIDS amp It was classified on the basis of the data from ECETOC Technical report No 48 2 P Positive N Negative 95 7 6 77 MATREX 77 MATREXTM Table 14 No 9 Maneb 3 LDM 46 6 63 3 f
86. 59 Sodium naphthalenesulfonate 0 0 P P True positive 60 Sodium stearate 2 4 N False positive 61 Sorbitan oleate 63 3 N 71 8 N N N True negative 62 Sorbitan sesquioleate 58 9 N 85 8 N N N True negative 63 Squalane 69 2 True negative 64 Stearalkonium chloride 47 True positive 65 TEA Lauryl sulfate 40 solution 1 4 True positive 66 Triacetin 6 8 N False positive 67 Triethylene glycol 77 0 N N N True negative 68 Triisopropanolamine 2 0 P True positive 69 Acetyl tributyl citrate 127 3 N N N True negative 70 Cetyl palmitate 47 2 47 5 N False 71 Decyl oleate 106 4 N N N True negative 72 Ethylhexyl stearate 91 0 N N N True negative 73 Glyceryl stearate 101 3 N N N True negative 74 PEG 2 stearate 85 3 N N N True negative 75 Castor seed oil 99 9 N N N True negative 76 Stearyl alcohol 82 0 N N N True negative 77 Zinc stearate 106 3 N N N True negative 2 n Dodecylthio ethanol and iso Octylthioglycolate were excluded from analysis due to the interference of the MTT assay P Positive N Negative NE Could not be evaluated Blank column NT Not tested 8 MATREX MATREX 34 7 1IC50
87. 829 6 Polyoxyethylene sorbitan monooleate 20E O 9005 65 6 Surfactants Liqui Calcium thioglycolate 814 71 1 Organic salts Soli 130 2 Distearyldimethylammonium chloride 107 64 2 Surfactants Soli 586 5 2 Ethylhexyl p dimethylamino benzonate 21245 02 3 PABA derivatives Liqui 277 4 Cetylpyridinium chloride 123 03 5 Surfactants Soli 340 Methyl p hydroxybenzoate 99 76 3 Esters Soli 152 2 Isopropyl myristate 110 27 0 Esters Liqui 270 5 Polyethylene glycol 400 25322 68 3 Polyols Liqui 360 400 Silicic anhydride 7631 86 9 Inorganics Soli 60 1 Benzyl alcohol 100 51 6 Alcohols Liqui 08 1 Sodium salicylate 54 21 7 Organic salts Soli 60 1 m Phenylenediamine 108 45 2 Amines Soli 08 1 Ethanol 64 17 5 Alcohols Liqui 64 1 Monoethanolamine 141 43 5 Alkanolamines Liqui 61 1 Triethanolamine 102 71 6 Alkanolamines Liqui 49 2 Stearyltrimethylammonium chloride 112 03 8 Surfactants Solid 348 1 Diisopropanolamine 110 97 4 Alkanolamines Solid 33 2 Potassium laurate 10124 65 9 Surfactants Solid 238 4 Cetyltrimethylammonium bromide 57 09 0 Surfactants Solid 364 5 Acetic aci 64 19 7 Carboxylic acids Liquid 60 1 Butanol 71 36 3 Alcohols Liquid 74 1 Chlorhexidine gluconate 20 solution 18472 51 0 Organic salts Solution 897 8 Domiphen bromide 538 71 6 Surfactants Solid 414 5 Lactic acid 50 21 5 Carboxylic acids Liquid 90 1 Glycolic acid 79 14 1 Carboxylic acids Solid 76 1 Di 2 ethylhexyl sodium sulfosuccinate 577 11 7 Surfactants Solid 488 5 MW Mo
88. P Glycerin N 5100 5100 5100 5100 5100 5100 5100 N N N N N N N 5100 5100 5100 5100 5100 5100 5100 N N N N N N N Polyoxyethylene N 1 5 Around 5 1 2 5 1 2 5 Around 2 5 7 5 10 2 5 5 P P P P P P P sorbitan 4 8 2 53 1 65 14 1 93 32 1 55 monooleate 20E O P P P P P P P Methyl N 1 5 Around 1 1 2 5 1 2 5 1 2 5 1 2 5 Around 2 p hydroxybenzoate 1 6 1 66 1 6 1 4 1 36 1 75 2 2 Sucrose fatty 0 025 0 01 0 025 0 05 Around 0 025 gt 0 01 0 01 0 02 0 01 0 025 0 01 0 025 P P NC P P P acid ester 0 027 0 014 0 024 0 009 0 013 0 02 0 033 P P Benzyl alcohol Around 7 5 Around 5 5 7 5 5 7 5 Around 7 5 7 5 10 Around 5 P P 74 7 8 6 6 2 8 2 7 15 64 Acid red 92 P Around 0 0075 Around 0 0075 0 0075 0 01 lt 0 005 0 005 0 01 lt 0 001 0 0075 0 01 0 0086 0 0062 0 0074 0 0038 0 0073 0 0008 0 018 Calcium 1 25 2 5 Around 5 Around 10 Around 7 5 1 2 5 7 5 10 1 2 Thioglycolate 1 4 6 4 6 7 7 2 15 7 1 5 m Phenylenediamine 0 25 0 5 5 1 5 0 25 0 5 lt 1 0 25 0 5 1 10 0 25 0 5 0 56 3 4 0 145 0 72 0 47 0 45 0 4 Sodium salicylate Around 7 5 10 2
89. P P P P P P P p hydroxybenzoate P P P P P Sucrose fatty P P P NC P P P acid ester P alcohol P P P P P Acid red 92 P P P P P Calcium P P thioglycolate P IP m Phenylenediamine B B P B P P P P P Sodium salicylate P P P Distearyldimethylammonium P P P P P chloride Cetylpyridinium chloride P P P P P P The in vivo results of as is application was predicted from the data of 10 concentration The repeated data were taken from range finding test upper column and main test lower column in the Japanese validation study The in vitro results were classified on the basis of their ICSO data m Phenylenediamine was excluded from analysis due to instability 17 P Positive N Negative Could not be classified 18 6 6 2 39 MATREX MATREX 50 N 50 P ART 50
90. Table 13 1 13 2 F No 287 77 A ERA 27 No 1 27 50 TJ z Database SIDS Screening Informa a IUCLID SID nd Toxicology of Chemicals MAS H7 IUCLID International Uniform ChemicaL Inforn nation tion Data Set ECETOC European Centre for Ecotoxicology y m EN S ARR du MAS15 NI Not Non irritati LAY Hi Sj AD REO A ng Slightly irritating MASIS NI GHS
91. of an in vitro tier system for the identification of cosmetic ingredients that are not ocular irritants Alternatives to Laboratory Animals 38 139 52 ICCVAM 2006 ICCVAM test method evaluation report Appendix H ICCVAM recommended reference substances list IUCLID http ecb jrc ec europa eu esis index php PGM dat Kay J H and Calandra J C 1962 Interpretation of eye irritation tests Journal of the society of cosmetic chemists 13 281 289 OECD OECD guideline for the testing of chemicals 405 Acute Eye Irritation Corrosion 2002 Ohno Y Kaneko T Inoue T Morikawa Y Yoshida T Fujii Masuda M Ohno Hayashi M Momma J Uchiyama T Chiba K Ikeda N Imanishi Y Itagaki H Kakishima H Kasai Y Kurishita A Kojima H Matsukawa K Nakamura T Ohkoshi K Okumura Saijo K Sakamoto K Suzuki T Takano K Tatsumi H Tani N Usami M and Watanabe R 1999 Interlaboratory validation of the 77 vitro eye irritation tests for cosmetic ingredients 1 Overview of the validation study and Draize scores for the evaluation of the tests Toxicology in Vitro 13 73 98 Ohno Y 2004 The validation and regulatory acceptance of alternative methods in Japan Alternatives to Laboratory Animals 32 Supplement 1 643 655 Ohuchi J Kasai Y Sakamoto Ohnuma M Kitamura M Kawasaki Y Kakishima Suzuki K Kuwahara H Imanishi Y Tatsumi H Kotani
92. tetrafluoroborate Sesame oil 3 Methoxy 1 2 propanediol Sorbitan oleate Toluene Sorbitan sesquioleate Squalane Cetyl alcohol Triethylene glycol Diisopropyl adipate Acetyl tributyl citrate PEG 40 stearate Decyloleate Sodium dehydroacetate Ethylhexyl stearate Sodium stearate Glyceryl stearate Triacetin PEG 2 stearate Castor seed oil Stearyl alcohol Zinc stearate The in vivo results of as is application was predicted from the data of 10 concentration m Phenylenediamine was excluded from the table due to instability 2 n Dodecylthio ethanol and iso octylthioglycolate were excluded from the table due to the interference for MTT assay 32 _ Table 18 Predictive capacity of the LDM MTT assay in the case of considering the equivocal classification N Sensitivity Specificity False positive False negative Concordance rate rate LDM MTT assay VS 102 100 54 46 0 80 Draize eye 56 56 25 46 21 46 0 56 81 102 test 9 MATREXTV Fan ETE TCR EO BEBUPE23 6 Draize MAS15
93. 0 solution P P 25 Butanol P P 26 Potassium laurate P P 27 Polyoxyethylene octylphenylether 10 E O P P 28 Di 2 ethylhexyl sodium sulfosuccinate P P 29 Acetic acid P P 30 Cetyltrimethylammonium bromide P P 31 Benzalkonium chloride P P 32 Stearyltrimethylammonium chloride P P 33 Cetylpyridinium chloride P P 34 Domiphen bromide P P reduction was evaluated as positive 57 The color of the MTT medium changed to blue purple significantly and the MTT The color of the MTT medium or the substance changed slightly The color of the MTT medium did not change and the MTT reduction was evaluated as negative NT not tested Red The color of the MTT medium changed red because acid red 92 was red dye 3 77 PROTA RGR 77 MTT table 2 1 2 2 Hexyl cinnamic aldehyde 12 5 Maneb 2 n Dodecylthio ethanol iso Octylthioglycolate Chlorophene Chloroxylenol Lauramide DEA Phenethyl alcohol Resorcinol 9 Hexyl cinnamic aldehyde 12 5 Maneb Chlorophene Chloroxylenol Lauramide DEA Phenethyl alcohol Resorcinol 7 MATREX Y
94. 02 05 06 08 10 11 2 3 5 7 9 10 12 3 5 7 10 12 2 4 7 9 11 01 02 08 04 06 07 08 Manufacturing month 6 MATREX 6 1 1991 12 1 MATREX GHS NI 50 EPEA CE m O ZZ ATE uu LEED YF MATREX vC H 6 2 Fig 7
95. 3 or2A gt 1 Benzyl alcohol 31 0 or2A 12 Ethanol 32 7 or2A gt 13 Acid red 92 71 0 or2A 14 Calcium thioglycolate 79 7 1 P 15 m Phenylenediamine 80 7 or2A 2 16 Sodium salicylate 83 7 or2A 17 Distearyldimethylammonium chloride 96 3 1 18 Lactic acid 102 7 1 1 100 gt 19 Sodium dodecyl sulfate 15 08 or2A NI 3 gt 20 Diisopropanolamine 23 0 1 2Aor2B gt 21 Monoethanolamine 23 3 or2A gt 22 Glycolic 25 0 or2A gt 23 Sodium hydrogenated tallow L glutamate 26 75 or2A P 24 Chlorhexidine gluconate 20 solution 28 3 or2A D 25 Butanol 34 0 or2A 1 10 gt 26 Potassium laurate 38 0 or2A 1 10 gt 27 Polyoxyethylene octylphenylether 10 E O 41 35 or2A 1 100 P 28 Di 2 ethylhexyl sodium sulfosuccinate 57 0 or2A 1 109 gt 29 Acetic acid 68 0 or2A 1 10 gt 30 Cetyltrimethylammonium bromide 76 75 or2A 1 109 P 31 Benzalkonium chloride 78 0 or2A 1 5 P 32 Stearyltrimethylammonium chloride 91 3 lt 1 P 33 Cetylpyridinium chloride 94 7 1 2 34 Domiphen bromide 96 3 or2A 1 109 gt The in vivo results of as is application were predicted from the data of 10 concentration Tested concentration is shown P Positive N Negative in parenthesis 16 6 6 MATREX IC50
96. 38 139 52 MATREX http www bio toyobo co jp bio01 file TMLDM 001 pdf OECD OECD guide skin irritation Recon ine for the testing of chemicals Draft proposal for a new guideline In vitro structed human epidermis RhE test method 2009 Ohuchi J Kasai Y Sakamoto K Ohnuma M Kitamura M Kawasaki Y Kakishima H Suzuki K Kuwahara H Imanishi Y Tatsumi H Kotani M Inoue K Okumura H Arashima M Kurishita A Kinoshita S Tani N Kojima H Nakamura T Suzuki K Ishibashi T Hori H Takahashi H Nishikawa T Kitano Y amp Ohno Y 1999 Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients 6 Evaluation of MATREX Toxicology in Vitro 13 153 162 1995 1 58 59 2008 SIRC 3 21 67 AT Jp 1 609 14 3 2 6484 52 2
97. 5 7 5 10 5 7 5 1 10 5 7 5 5 10 9 2 9 8 8 5 6 5 47 11 5 11 5 P P Distearyldimethylammonium 0 1 0 5 5 7 5 0 1 0 25 lt 0 05 0 01 0 025 0 25 0 75 0 10 Chloride 0 11 0 072 0 295 0 092 0 0125 0 22 0 07 Cetylpyridinium chloride 0 001 0 0075 0 001 0 0025 0 001 0 0025 Around 0 001 lt 0 001 0 001 0 0025 0 001 0 0025 0 0027 0 00133 0 00265 0 0013 0 00124 0 00165 0 0026 The in vivo results of as is application was predicted from the data of 10 concentration The repeated data were taken from range finding test upper column and main test lower column in the Japanese validation study P Positive N Negative NC Could not be classified 51 Appendix 9 MTT MTT 4100 LDM MTT MTT LDM or LOM eS Jp 1 MTT
98. 76 Stearyl alcohol 82 0 N N N N True negative 77 Zinc stearate 106 3 N N N N True negative P Positive N Negative NE Could not be evaluated Blank column NT Not tested 55 Appendix 10 5 MTT 5 MTT Appendix 9 5 MTT 5 MATREX 1 Table 1 Results of Interference of MTT assay 5 substances evaluated as negative in the Japanese validation study Substance The previous results in the Japanese validation study In vitro In vivo Evaluation Lab Lab Lab Lab Lab Lab Lab Final popsa Results Classifica A B C D E F G results tion 2 Ethylhexyl N N N N N N N N N N True negative p dimethylamino benzoate Isopropyl N N N N N N N N N N True negative myristat
99. D Table 1 The untreated control data in the LDM MTT assay The date of the measurement 0 387 February 11 2010 0 372 March 11 2010 0 308 March 11 2010 0 398 March 11 2010 0 343 March 11 2010 0 379 March 11 2010 0 376 March 11 2010 0 366 March 11 2010 0 362 March 11 2010 0 354 March 11 2010 0 440 26 2010 0 359 March 26 2010 0 376 March 26 2010 0 371 26 2010 0 358 March 26 2010 0 393 March 26 2010 0 370 26 2010 0 368 March 26 2010 0 367 March 26 2010 0 360 March 26 2010 0 352 March 26 2010 0 405 March 26 2010 0 427 March 26 2010 0 390 March 26 2010 0 427 March 26 2010 0 381 March 26 2010 0 337 April 8 2010 0 313 April 8 2010 0 307 April 8 2010 0 310 April 8 2010 0 309 June 3 2010 0 271 June 3 2010 0 255 June 3 2010 0 245 June 3 2010 Unacceptable 0 300 June 3 2010 0 296 June 3 2010 0 273 June 3 2010 0 295 June 3 2010 0 293 June 3 2010 0 365 June 17 2010 49 2 SDS Table 2 The data of 0 01 and 0 1 SDS in the LDM MTT assay OD The dat
100. ECETOC GHS NI N P Table 13 1 GHS classification of the substances GHS classification by Substance ICCVAM Recommended In vivo Reference Substance List 1 Ethyl 2 methyl acetoacetate 2B P 2 Ammonium nitrate 2B P 3 Butyl Dipropasol Solvent 2B P 4 3 Chloropropionitrile 2B P 5 Cyclopentanol 2B P 6 3 3 Dithiodipropionic acid 2B P 7 Hexyl cinnamic aldehyde 2B 12 595 8 N Lauroylsarcosine sodium salt 2B P 9 Maneb 2B 10 2 Methyl 1 pentanol 2B P 11 Propasol Solvent P 2B P 12 6 Methyl purine 2B P 13 2 6 Dichloro 5 fluoro beta oxo 3 pyridinepropanoate 2B P 14 Triton X 100 1 100 2B 5 NI 1 15 iso Octyl acrylate NI N 16 tetra Aminopyrimidine sulfate NI N 17 2 4 Difluoronitrobenzene NI N 18 n n Dimethylguanidine sulfate NI N 19 2 n Dodecylthio ethanol NI N 20 iso Propyl bromide NI N 21 Di iso butyl ketone NI N 22 iso Octylthioglycolate NI N 23 2 4 Pentanediol NI N 24 2 2 Dimethyl 3 pentanol NI N 25 Potassium tetrafluoroborate NI N 26 3 Methoxy 1 2 propanediol NI N 27 Toluene NI N Tested concentration is shown in parenthesis P Positive N Negative 94 Table 13 2 GHS classi
101. PC H HZ CX BSA Hx gv loride CPC CAE H CPC DEB MTT Fig 1 ML KY H E HL 1 CPC HL7 1 CPC 1006 4 16CPC 0 1 MTT MTT
102. T CVS 1 000 0 969 1 000 0 370 0 331 Non irritants for which IC50 values are not determined are given maximum values 39 EYTEX 1 000 Appendix 3 MATREX MATREX E 2 DMEM Dulbecco s Modified Eagle Medium MATREX 20 30 16 19 Fig 1 Fig 1 MATREX 1688 19 ae 3 1 1 LY 4 20 3055 40
103. acroscopic inspection of Adequate Inadequate 5 4 OD LDM OD 216 0 385 0 065 LDM OD 0 30 216 Fig 5 Appendix 5 Fig 5 OD of untreated control in the LDM MTT assay 0 800 0 700 0 600 0 500 0 400 0 300 0 200 0 100 0 000 9 123 5 6 9 11 01 02 05 06 08 10 11 2 3 5 7 9 10 12 3 5 7 10122 4 7 9 11 01 2 08 04 06 07 08 Manufacturing month 5 5 12050 CaS Sodium dodecyl sulfate SLS IC50 216 0 016 0 002 SDS 1050 0 010 0 020 216 Fig 6 Appendix 5 Fig 6 IC50 of positive control SDS in the LDM MTTassay IC50 99 of SDS 0 025 0 020 0 015 0 010 9 12 3 5 9 11 01
104. aurate 10 E O classified 38 Sodium N lauryl sarcosinate Could not be 30 solution classified 39 Polyoxyethylene hydrogenated Could not be P P P castor oil 60 E O classified The in vivo results of as is application was predicted from the data of 10 concentration M Phenylenediamine was excluded from analysis due to instability P Positive N Negative Blank column NT Not tested 20 7 MATREX 7 1 F 2 1991 4EBE ZX ELPA SHEED 12 MATREX GHS NI GHS NI 9 NI ICCVAM Recommended E ED FA
105. cted from the data of 0 1 solution as a positive control P Positive N Negative Blank column Not tested 62 Appendix 14 zn vivo Table 1 References of the in vivo data of the 50 substances No Substance References 28 2 Bromo 2 Nitropropane 1 3 Diol 3 3 139 155 1984 JEPT 4 4 47 61 1980 29 Benzalkonium chloride JACT 8 4 589 625 1989 30 Benzophenone 1 2 5 35 77 1983 31 Benzophenone 2 JACT 2 5 79 84 1983 32 Butoxyethanol 15 6 462 526 1996 33 Butylene glycol Hifu 26 5 1065 1074 1984 34 Cetrimonium chloride IT 16 S3 195 220 1997 35 Cetyl alcohol JACT 7 3 359 413 1988 36 Chlorhexidine digluconate 2096 Solution JACT 12 3 201 23 1993 37 Chlorophene 23 S1 1 27 2004 38 Chloroxylenol JACT 4 5 147 69 1985 39 Diethylhexyl adipate JACT 3 3 101 30 1984 40 Diisopropyl adipate JACT 3 3 101 30 1984 41 Dioctyl sodium sulfosuccinate 17 S4 1 20 1998 42 palmitate JACT 1 2 13 35 1982 43 Hexylene glycol JACT 4 5 223 48 1985 44 Isocetyl stearate JACT 4 5 107 46 1985 45 Isopropyl Myristate JACT 1 4 55 80 1982 46 Isopropyl Palmitate JACT 1 2 13 35 1982 47 Lauramide DEA JACT 5 5 415 54 1986 48 Methoxyisopropyl acetate
106. e Di 2 ethylhexyl sodium sulfosuccinate Dioctyl sodium sulfosuccinate 5 Isopropyl myristate 4 GHS NI 1 4 1 2 1 Table 1 The comparison between the Japanese validation study data and the additional data In vivo The Japanese validation study data Substance a classification Lab A Lab B Lab C Lab D LabE Lab F Lab G Lab H Isopropyl N N N N N N N N N myristate Sodium dodecyl P P P sulfate Triton X 100 Di 2 ethylhexyl P P P P sodium sulfosuccinate Benzalkonium P P P P P chloride The in vivo results of as is application was predicted from the data of 10 concentration The results of the sodium dodecyl sulfate are predi
107. e Isotonic sodium N N N N N N N True negative chloride solution Silicic anhydride N N N N N N N N N N True negative Glycerin N N N N N N N N N N True negative P Positive N Negative Blank column Not tested 56 Appendix 11 MTT T 1 80 LEIE 37 3 MTT MTT 2 34 34 MTT Calcium thioglycolate 34 E Ex Table 1 m Phenylenediamine Diisopropanolamine H 77 MTT XJ 80mg 0 333mg mLMTT 1 2mL MTT
108. e of the 0 01 0 1 measurement SDS SDS 0 605 0 054 February 11 2010 0 331 0 010 March 11 2010 0 330 0 013 March 11 2010 0 422 0 010 March 11 2010 0 386 0 006 March 11 2010 0 371 0 011 March 11 2010 0 424 0 013 March 11 2010 0 334 0 013 March 11 2010 0 366 0 013 March 11 2010 0 411 0 012 March 11 2010 0 482 0 009 March 26 2010 0 345 0 022 March 26 2010 0 424 0 020 March 26 2010 0 378 0 014 March 26 2010 0 425 0 008 March 26 2010 0 399 0 012 March 26 2010 0 406 0 014 March 26 2010 0 502 0 016 March 26 2010 0 418 0 024 26 2010 0 380 0 010 March 26 2010 0 441 0 009 March 26 2010 0 514 0 015 March 26 2010 0 431 0 008 March 26 2010 0 481 0 006 March 26 2010 0 434 0 009 March 26 2010 0 405 0 008 March 26 2010 0 407 0 011 April 8 2010 0 431 0 011 April 8 2010 0 389 0 013 April 8 2010 0 397 0 012 April 8 2010 0 366 0 009 June 3 2010 The SDS was obtained from Wako Pure Chemical Industries Ltd Lot TCG8194 Appendix 8 7 CER AST DICER DE 7 gt tras PN 2 21 au ui 7 AERO 2 2 2
109. fication of the substances GHS GHS classification classification by ICCVAM mE 05280Om obtained GHS classification obtained from In vivo Substance Recommended predicted from the from Reference previous paper Japancs grobal chemical databases classification Substance List validation study 28 2 Bromo 2 Nitropropane 1 3 Diol 2B 2A or 29 Benzalkonium chloride 1 5 2B 2A or 1 2Aor1 1 10 P 10 30 Benzophenone 1 2B P 31 Benzophenone 2 2B P 32 Butoxyethanol 1 2B or2A 2B or 2A 33 Butylene glycol NI 34 Cetrimonium chloride 2B 2A or 1 P 35 Cetyl alcohol NI N 36 Chlorhexidine digluconate 2Aor1 P 37 Chlorophene 2Aorl 38 Chloroxylenol 2B 2A or 1 P 39 Diethylhexyl adipate NI NF NP N 40 Diisopropyl adipate NI N 41 Dioctyl sodium sulfosuccinate 2B 2A or 1 2A P 42 Ethylhexyl palmitate NI N 43 Hexylene glycol 2B 2A or 2B 2A 1 NL2Bor2A 44 Isocetyl stearate NI N 45 Isopropyl Myristate NI NI 46 Isopropyl Palmitate NI N 47 Lauramide DEA 2A or 1 48 Methoxyisopropyl acetate 2B 2A or N 2B or 2A 49 Oleyl alcohol NI N 50 PEG 40 stearate NI N 51 alcohol 2B 2A 1 2B or 24 P 52 Phenoxyethanol 2A 2B or 2B 2A 15 P 53 Phytantriol 2B 2A or P 54 Propylene carbonate NI 2B or 2A 55 Resorcinol 2B 2A or
110. hydroacetate N P 59 Sodium naphthalenesulfonate P P ae 60 Sodium stearate N 61 Sorbitan oleate N N 62 Sorbitan sesquioleate N N 63 Squalane N N 64 Stearalkonium chloride P P 65 TEA Lauryl sulfate 4096 solution P P 66 Triacetin N P 67 Triethylene glycol N N 68 Triisopropanolamine P P 69 Acetyl tributyl citrate N N 70 Cetyl palmitate N P 7l Decyl oleate N N 72 Ethylhexyl stearate N N 73 Glyceryl stearate N N 74 PEG 2 stearate N N 75 Castor seed oil N N 76 Stearyl alcohol N N 77 Zinc stearate N N The color of the MTT medium changed to blue purple significantly and the MTT reduction was evaluated as positive The color of the MTT medium or the substance changed slightly The color of the MTT medium did not change and the MTT reduction was evaluated as negative P Positive N Negative 59 Appendix 12 50 2008 4 H iH 21 LDM MTT JSAAE 50 49
111. il 8008 74 0 Oils Liquid 58 Sodium dehydroacetate 4418 26 2 Organic salts Solid 90 1 59 Sodium naphthalenesulfonate 532 02 5 Organic salts Solid 230 2 60 Sodium stearate 822 16 2 Surfactants Solid 306 5 61 Sorbitan oleate 1338 43 8 Surfactants Liquid 428 6 62 Sorbitan sesquioleate 8007 43 0 Surfactants Liquid 1175 7 63 Squalane 111 01 3 Hydrocarbons Liquid 422 8 64 Stearalkonium chloride 122 19 0 Surfactants Solid 424 2 65 TEA Lauryl sulfate 40 solution 139 96 8 Surfactants Solution 415 6 66 Triacetin 102 76 1 Triacetates Liquid 218 2 67 Triethylene glycol 112 27 6 Polyols Liquid 150 2 68 Triisopropanolamine 122 20 3 Amines Solid 191 3 23 69 Acetyl tributyl citrate 77 90 7 Esters Liquid 402 5 70 Cetyl palmitate 540 10 3 Esters Solid 480 9 71 Decyl oleate 3687 46 5 Esters Liquid 422 7 72 Ethylhexyl stearate 22047 49 0 Esters Liquid 396 7 73 Glyceryl stearate 11099 07 3 Esters Solid 358 6 74 PEG 2 stearate 106 11 6 Surfactants Solid 89 1 75 Castor seed oil 8001 79 4 Oils Liquid 76 Stearyl alcohol 112 92 5 Alcohols Solid 270 5 77 Zinc stearate 557 05 1 Organic metals Solid 632 3 MW Molecular weight 7 5 Draize ICCVAM Recommended Reference Substance List GHS
112. kedly deepened rugae congestion swelling moderate circumcorneal hyperaemia or injection iris reactive to light a sluggish reaction is considered to be an effect Hemorrhage gross destruction or no reaction to light Maximum possible 2 Conjunctivae Redness refers to palpebral and bulbar conjunctivae excluding cornea and iris Some blood vessels hyperaemic injected Diffuse crimson colour individual vessels not easily discernible Diffuse beefy red Maximum possible 3 Chemosis Swelling refers to lids and or nictating membranes Normal Some swelling above norma Obvious swelling with partial eversion of lids Swelling with lids about half closed Swelling with lids more than half closed Maximum possible 4 table is the same as that of OECD guideline for the testing of chemicals 405 Acute eye irritation Corrosion 2002 Table 2 GHS classification of serious eye damage eye irritation Caterory Criteria of GHS a at least in one animal effects on the cornea iris or conjunctiva that b at least in 2 of 3 tested animals a positive response of i corneal opacity gt 3 and or ii iritis gt 1 5 test material 1 An eye irritant Category 1 irreversible effects on the eye is a test material that produces have not fully reversed within an observation period of normally 21 days and or calculated as the mean socres following grading at 24 48 and 72 hours after installation of
113. l 1979 1987 Organogenesis Inc 1994 US4 539 716 US4 546 500 207 7 v E CE E a 5 53505 1993 8 10 2008 8 H 10 Organogenesis Inc C452 TES 26 DMEM Dulbecco s Modified Eagle Medium MATREX 16 DEAE 5
114. lecular weight 14 6 5 Draize Draize Table 7 GHS GNI 9 1 of the GHS HA P Positive 2A 2B lt 9 GHS the third revised edition GHS 1or2A Draize 14 21 1 2A NI 1or2A GHS NI N Negative Table 7 Draize eye test results in the Japanese validation study as is
115. lene carbonate N P P 55 Resorcinol P P P 56 Safflower oil N N N 57 Sesame oil N N N 58 Sodium dehydroacetate N P P 59 Sodium naphthalenesulfonate P P P 60 Sodium stearate N P P 61 Sorbitan oleate N N N 62 Sorbitan sesquioleate N N N 63 Squalane N N N 64 Stearalkonium chloride P P P 65 TEA Lauryl sulfate 40 Solution 66 Triacetin N P P 67 Triethylene glycol N N N 68 Triisopropanolamine P P P 69 Acetyl tributyl citrate N N N 70 Cetyl palmitate N P P 71 Decyl oleate N N N 72 Ethylhexyl stearate N N N 73 Glyceryl stearate N N N 74 PBG 2 stearate N N N 75 Castor seed oil N N N 76 Stearyl alcohol N N N 77 Zinc stearate N N N P Positive N Negative 60 Table 2 Results of phytantriol in the LDM MTT assay Viablity Additional data JSAAE data First measurement 58 1 28 2 Negative Positive Second measurement 55 2 42 7 Negative Positive Final result Negative Positive 61 vi Appendix 13 1 2 Isopropyl myristate Polyoxyethylene octylphenylether 10 51 Triton X 100 Sodium dodecyl sulfate Benzalkonium chlorid
116. lthio ethanol iso Propyl bromide Di iso butyl ketone 2 4 Pentanediol 2 2 Dimethyl 3 pentanol Potassium tetrafluoroborate 3 Methoxy 1 2 propanediol Toluene Cetyl alcohol Diisopropyl adipate PEG 40 stearate Sodium dehydroacetate Sodium stearate Triacetin Cetyl palmitate 9856 57 53 25 47 47 22 47 2 1 57 78 81 104 Table 16 Phytantriol 50 JSAAE 50 Equivocal 40 60 Equivocal Table 17 3 Equivocal 1 E2528 I8 E2388 RE 100 56 56 54 25 46 46 21 46 0 0 56 79 81 102 Table 18 MATREX
117. mmonium bromide Benzalkonium chloride Stearyltrimethylammonium chloride Cetylpyridinium chloride Domiphen bromide Ethyl 2 methyl acetoacetate Ammonium nitrate Butyl Dipropasol Solvent 3 Chloropropionitrile Cyclopentanol 3 3 Dithiodipropionic acid Hexyl cinnamic aldehyde 12 5 N Lauroylsarcosine sodium salt Maneb 2 Methyl 1 pentanol Propasol Solvent P 6 Methyl purine 2 6 Dichloro 5 fluoro beta oxo 3 pyridinepropanoate 2 Bromo 2 Nitropropane 1 3 Diol Benzophenone 1 Benzophenone 2 Butoxyethanol Cetrimonium chloride Chlorhexidine digluconate Chlorophene Chloroxylenol Hexylene glycol Lauramide DEA Methoxyisopropyl acetate Phenethyl alcohol Phenoxyethanol Propylene carbonate Resorcinol Sodium naphthalenesulfonate Stearalkonium chloride TEA Lauryl sulfate 40 solution Triisopropanolamine Negative 21 25 NI Polyethylene glycol 400 Cetyl palmitate 2 Ethylhexyl p dimethylamino benzoate Polyoxyethylene sorbitan monooleate 20E O Isopropyl myristate Triethanolamine Isotonic sodium chloride solution Methyl p hydroxybenzoate Silicic anhydride Glycerin iso Octyl acrylate tetra Aminopyrimidine sulfate Butylene glycol 2 4 Difluoronitrobenzene Diethylhexyl adipate n n Dimethylguanidine sulfate Ethylhexyl palmitate iso Propyl bromide Isocetyl stearate Di iso butyl ketone Isopropyl Palmitate 2 4 Pentanediol Oleylalcoho 2 2 Dimethyl 3 pentanol Safflower oi Potassium
118. olid 200 0 29 Benzalkonium chloride 8001 54 5 Surfactants Solid 283 9 30 Benzophenone 1 31 56 6 Benzophenones Solid 214 2 31 Benzophenone 2 31 55 5 Benzophenones Solid 246 2 32 Butoxyethanol 11 76 2 Alcohols Liquid 118 2 33 Butylene glycol 07 88 0 Polyols Liquid 90 1 34 Cetrimonium chloride 12 02 7 Surfactants Solid 320 0 35 Cetyl alcohol 36653 82 4 Alcohols Solid 242 4 36 Chlorhexidine digluconate 20 solution 18472 51 0 Organic salts Solution 897 8 37 Chlorophene 20 32 1 Aromatics Solid 218 7 38 Chloroxylenol 88 04 0 Aromatics Solid 156 6 39 Diethylhexyl adipate 03 23 1 Esters Liquid 370 6 40 Diisopropyl adipate 6938 94 9 Esters Liquid 230 3 41 Dioctyl sodium sulfosuccinate 577 11 7 Surfactants Solid 488 5 42 Ethylhexyl palmitate 29806 73 3 Esters Liquid 368 6 43 Hexylene glycol 07 41 5 Polyols Liquid 118 2 44 Isocetyl stearate 25339 09 7 Esters Liquid 508 9 45 Isopropyl Myristate 10 27 0 Esters Liquid 270 45 46 Isopropyl Palmitate 42 91 6 Esters Liquid 298 5 47 Lauramide DEA 20 40 1 Surfactants Solid 287 4 48 Methoxyisopropyl acetate 08 65 6 Esters Liquid 32 2 49 Oley alcohol 43 28 2 Alcohols Liquid 268 5 50 PEG 40 stearate 9004 99 3 Surfactants Solid 51 Phenethyl alcohol 60 12 8 Alcohols Liquid 22 2 52 Phenoxyethanol 22 99 6 Alcohols Liquid 38 2 53 Phytantriol 74563 64 7 Polyols Liquid 330 6 54 Propylene carbonate 08 32 7 Dioxolanes Liquid 02 1 55 Resorcinol 08 46 3 Aromatics Solid 10 1 56 Safflower oil 8001 23 8 Oils Liquid 57 Sesame o
119. olvent 29911 27 1 Ethers Liquid 176 3 4 3 Chloropropionitrile 542 76 7 Nitriles Liquid 89 5 5 Cyclopentanol 96 41 3 Alcohols Liquid 86 1 6 3 3 Dithiodipropionic acid 1119 62 6 Acids Solid 210 3 7 Hexyl cinnamic aldehyde 101 86 0 Aldehydes Liquid 216 3 8 N Lauroylsarcosine sodium salt 137 16 6 Surfactants Solid 293 4 9 Maneb 12427 38 2 Organic metals Solid 265 3 10 2 Methyl 1 pentanol 105 30 6 Alcohols Liquid 02 2 11 Propasol Solvent P 1569 01 3 Ethers Liquid 18 2 12 6 Methyl purine 2004 03 7 Heterocyclic compounds Solid 34 13 2 6 Dichloro 5 fluoro beta oxo 3 pyridinepropanoate 96568 04 6 Esters Solid 280 14 Triton X 100 9002 93 1 Surfactants Liquid 250 4 15 iso Octyl acrylate 29590 42 9 Esters Liquid 84 3 16 tetra Aminopyrimidine sulfate 5392 28 9 Organic salts Solid 238 2 17 2 4 Difluoronitrobenzene 446 35 5 Aromatics Liquid 59 18 n n Dimethyl guanidine sulfate 598 65 2 Organic salts Solid 272 3 19 2 n Dodecylthio ethanol 1462 55 1 Alcohols Liquid 206 3 20 iso Propyl bromide 75 26 3 Halogenated Liquid 23 0 hydrocarbon 21 Di iso butyl ketone 08 83 8 Ketones Liquid 42 2 22 iso Octylthioglycolate 25103 09 7 Thiols Liquid 204 3 23 2 4 Pentanediol 625 69 4 Polyols Liquid 04 2 24 2 2 Dimethyl 3 pentanol 3970 62 5 Alcohols Liquid 16 2 25 Potassium tetrafluoroborate 14075 53 7 Inorganic salts Solid 25 9 26 3 Methoxy 1 2 propanediol 623 39 2 Polyols Liquid 06 1 27 Toluene 08 88 3 Aromatics Liquid 92 1 28 2 Bromo 2 Nitropropane 1 3 Diol 52 51 7 Polyols S
120. oxicology JEPT Journal of Environmental Pathology amp Toxicology 68 Appendix 15 MATREX 77 2010 2 H 9 2 12 1 1 77 MATREX Y GHS N bo MATREX 24 MTT Bi
121. say MTT assay medium 54 3 77 MATREX 24 22 MTT 2 n Dodecylthio ethanol iso Octylthioglycolate 2 2 n Dodecylthio ethanol MTT MTT 0 1 44 6 MATREX iso Octylthioglycolate 56 3 30 MTT j gt Table 1 Results of the MATREX test included the interference data of the MTT assay
122. te Castor seed oil Stearyl alcohol Zinc stearate The in vivo results of as is application was predicted from the data of 10 concentration m Phenylenediamine was excluded from the table due to instability 2 n Dodecylthio ethanol and iso octylthioglycolate were excluded from the table due to the interference for MTT assay 30 Table 16 Predictive capacity of the LDM MTT assay N Sensitivity Specificity False positive False negative Concordance rate rate LDM MTT assay VS 104 98 53 47 2 78 Draize eye 56 57 25 47 23 47 1 57 81 104 test 31 Table 17 The eye irritancy of test samples predicted by LDM MTT assay in the case of considering the equivocal classification In vitro Classification by SIRC CVS assay using triethanolamine as a reference substance for non irritancy Positive Equivocal Negative In vivo Positive 56 1 0 Classification by 1 2Aor 2B Sucrose fatty acid ester Phytantriol GHS Benzyl alcohol Ethanol Acid red 92 Calcium thioglycolateSodium salicylate Distearyldimethylammonium chloride Lactic acid Sodium dodecyl sulfate Diisopropanolamine Monoethanolamine Glycolic acid Sodium hydrogenated tallow L glutamate Chlorhexidine gluconate 20 solution Butanol Potassium laurate Polyoxyethylene octylphenylether 10 E O Di 2 ethylhexyl sodium sulfosuccinate Acetic acid Cetyltrimethyla
123. the are not expected to reverse or a at least in 2 of 3 tested animals a positive response of i corneal opacity gt 1 and or ii iritis gt 1 and or iii conjunctival redness gt 2 and or iv conjunctival oedema chemosis gt 2 Within this category an eye irritant is considered mildly irritating to effects listed above are fully reversible within 7 days of observation 2 An eye irritant Category 2A irritating to eyes is a test material that produces calculated as the mean socres following grading at 24 48 and 72 hours after installation of the test material and which fully reverses within an observation period of normally 21 days eyes Category 2B when the The table is the same as the third revised edition of the GHS 2009 3 Appendix1 4 MATREX 2008 Xf 1996 Draize
124. ton Byaluation Negative N Negative N results 28 2 Bromo 2 Nitropropane 1 3 Diol 9 9 P P True positive 29 Benzalkonium chloride 5 0 True positive 30 Benzophenone 1 1 6 P True positive 31 Benzophenone 2 4 0 True positive 32 Butoxyethanol 2 5 True positive 33 Butylene glycol 71 8 N N N True negative 34 Cetrimonium chloride 5 0 True positive 35 Cetylalcohol 21 2 P N False positive 36 Chlorhexidine digluconate 20 41 True positive solution 37 Chlorophene 13 2 P P True positive 38 Chloroxylenol 3 3 True positive 39 Diethylhexyl adipate 107 1 N N N True negative 40 Diisopropyl adipate 2 9 N False positive 41 Dioctyl sodium sulfosuccinate 3 8 True positive 42 Ethylhexyl palmitate 71 2 N N N True negative 43 Hexylene glycol 2 2 True positive 44 Isocetyl stearate 100 9 N N N True negative 45 Isopropyl Myristate 98 4 N N N True negative 46 Isopropyl Palmitate 102 8 N N N True negative 47 Lauramide DEA 22 True positive 48 Methoxyisopropyl acetate 13 P P True positive 49 Oleylalcohol 89 0 N N N True negative 50 PEG 40 stearate 1 9 N False positive 51 Phenethyl alcohol 3 5 P P True positive 52 Phenoxyethanol 1 8 P P True positive 53 Phytantriol 58 1 N 55 2 N N P False negative 54 Propylene carbonate 4 8 True positive 55 Resorcinol 10 0 P P True positive 56 Safflower oil 104 9 N N N True negative 57 Sesame oil 101 3 N N N True negative 58 Sodium dehydroacetate 1 7 P N False positive
125. ution 4 Silicic anhydride N N N N N N N N 5 Polyethylene glycol 400 N N N P P N P P 6 Glycerin N N N N N N N N 7 Polyoxyethylene sorbitan N P P P P P P P monooleate 20E O 8 Triethanolamine N P P P P 9 Methyl p hydroxybenzoate N P P P P P P P 10 Sucrose fatty acid ester P P P P P P P P 11 Benzyl alcohol P P P P P P P P 12 Ethanol P P P P P 13 Acidred 92 P P P P P P P P 14 Calcium thioglycolate P P P P P P P P 15 m Phenylenediamine P P P P P P P P 16 Sodium salicylate P P P P P P P P 17 Distearyldimethylammonium P P P P P P P P chloride 18 Lactic acid P P P P P 19 Sodium dodecyl sulfate P P P P 20 Diisopropanolamine P P P P P 21 Monoethanolamine P P P P P 22 Glycolic acid P P P P P 23 Sodium hydrogenated tallow P P P P L glutamate 24 Chlorhexidine gluconate 2090 P P P P P solution 25 Butanol P P P P P 26 Potassium laurate P P P P P 27 Polyoxyethylene P P P P octylphenylether 10 E O 28 Di 2 ethylhexyl sodium P P P P P sulfosuccinate 29 Acetic acid P P P P P 30 Cetyltrimethylammonium P P P P P bromide 31 Benzalkonium chloride P P P P 32 Stearyltrimethylammonium P P P P P chloride 33 Cetylpyridinium chloride P P P P P P P P 34 Domiphen bromide P P P P P 35 Polyoxyethylene hydrogenated Could not be P P P castor oil 60 E O classified 36 Polyoxyethylene sorbitan Could not be P P P monolaurate 20 E O classified 37 Polyethyleneglycol Could not be P P P monol
126. was evaluated as negative 2 n Dodecylthio ethanol and iso Octylthioglycolate could not be evaluated due to the interference of the MTT assay However the uncorrected results of the MTT 58 assay were negative when the interference of the MTT was not considered P Positive N Negative NE Could not be evaluated Table 2 2 Results of the MTT reduction by the 77 substances In vivo No Substance Classifica In vitro results reduction 28 2 Bromo 2 Nitropropane 1 3 Diol 29 Benzalkonium chloride P P 30 Benzophenone 1 P P 31 Benzophenone 2 P P 32 Butoxyethanol 33 Butylene glycol N N 34 Cetrimonium chloride P P 35 Cetylalcohol N P 36 Chlorhexidine digluconate 2096 solution P P B7 Chlorophene P P ar 38 Chloroxylenol 39 Diethylhexyl adipate N N 40 Diisopropyl adipate 41 Dioctyl sodium sulfosuccinate P P 42 Ethylhexyl palmitate N N 43 Hexylene glycol P 44 Isocetyl stearate N N 45 Isopropyl myristate N N 46 Isopropyl Palmitate N N 47 Lauramide DEA P P 48 Methoxyisopropyl acetate P P 49 Oleyl alcohol N N 50 40 stearate N P 51 Phenethyl alcohol P P t 52 Phenoxyethanol P P 53 Phytantriol N 54 Propylene carbonate 55 Resorcinol P 56 Safflower oil N N ae 57 Sesame oil N N 58 Sodium de
127. x 19 40mg mL 1996 20 30 4 Appendix 3 1X107 cm 1 5 2 MATREX LDM Fig 1 LDM 1 200 u m CHS Fig 2 DMEM Ham E
128. ylsarcosine sodium salt Maneb 2 Methyl 1 pentanol Propasol Solvent P 6 Methyl purine 2 6 Dichloro 5 fluoro beta oxo 3 pyridinepropanoate 2 Bromo 2 Nitropropane 1 3 Diol Benzophenone 1 Benzophenone 2 Butoxyethanol Cetrimonium chloride Chlorhexidine digluconate Chlorophene Chloroxylenol Hexylene glycol Lauramide DEA Methoxyisopropyl acetate Phenethyl alcohol Phenoxyethanol Propylene carbonate Resorcinol Sodium naphthalenesulfonate Stearalkonium chloride TEA Lauryl sulfate 40 solution Triisopropanolamine Negative 22 25 NI Polyethylene glycol 400 2 Ethylhexyl p dimethylamino benzoate Polyoxyethylene sorbitan monooleate 20 Isopropyl myristate Triethanolamine Isotonic sodium chloride solution Methyl p hydroxybenzoate Silicic anhydride Glycerin iso Octyl acrylate tetra Aminopyrimidine sulfate Butylene glycol 2 4 Difluoronitrobenzene Diethylhexyl adipate n n Dimethylguanidine sulfate Ethylhexyl palmitate iso Propyl bromide Isocetyl stearate Di iso butyl ketone Isopropyl Palmitate 2 4 Pentanediol Oley alcoho 2 2 Dimethyl 3 pentanol Safflower oi Potassium tetrafluoroborate Sesame oil 3 Methoxy 1 2 propanediol Sorbitan oleate Toluene Sorbitan sesquioleate Squalane Cetyl alcohol Triethylene glycol Diisopropyl adipate Acetyl tributyl citrate PEG 40 stearate Decyloleate Sodium dehydroacetate Ethylhexyl stearate Sodium stearate Glyceryl stearate Triacetin PEG 2 stearate Cetyl palmita
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