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OECD OCDE 432 Draft
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432 OECD OCDE, 45 PRINCIPLE OF THE TEST METHOD, 47 7 The in vitro 3T3 NRU phototoxicity test is based on a comparison of the cytotoxicity of a. 48 chemical when tested in the presence and in the absence of exposure to a non cytotoxic dose of simulated. 49 solar light Cytotoxicity in this test is expressed as a concentration dependent reduction of the uptake of. 50 the vital dye Neutral Red NR when measured 18 24 hours after treatment with the test chemical and. 51 irradiation 13 NR is a weak cationic dye that readily penetrates cell membranes by non ionic diffusion. 52 and accumulates intracellularly in lysosomes NR is not charged at close to neutral pH of the cytoplasm. 53 but becomes positively charged and trapped in low pH of lysosomal lumen The low pH of lysosomal. 54 lumen is actively maintained requires ATP and is dependent on integrity of lysosomal membrane. 55 Phototoxins can induce cell damage through formation of Reactive Oxygen Species ROS and other. 56 mechanisms that lead to increased permeability of the lysosomal membrane reduction in the pH gradient. 57 and other changes that gradually become irreversible 214 15 Such changes brought about by the action. 58 of xenobiotics result in a decreased uptake and binding of NR It is thus possible to distinguish between. 59 viable and damaged or dead cells, 61 8 BALB c 3T3 cells are maintained in culture for 18 24 h for formation of monolayers Two 96. 62 well plates per test chemical are pre incubated with eight different concentrations of the test chemical for 1. 63 h Thereafter one of the two plates is exposed to an irradiation dose whereas the other plate is kept in the. 64 dark In both plates the treatment buffer is then replaced with culture medium and overnight incubation. 65 18 24 h cell viability is determined by NRU Cell viability is expressed as percentage of test chemical. 66 treated NRU values compared with solvent controls and is calculated for each test concentration To. 67 predict the phototoxic potential the concentration responses obtained in the presence and in the absence of. 68 irradiation are compared including the concentration reducing cell viability to 50 compared to the. 69 solvent controls i e IC50, 72 DESCRIPTION OF THE TEST METHOD. 74 Preparations, 78 9 A immortalised mouse fibroblast cell line BALB c 3T3 clone A31 obtained from either from. 79 the American Type Culture Collection ATCC Manassas VA USA or from the European Collection of. 80 Cell Cultures ECACC Salisbury Wiltshire UK was used in the validation study It is recommended. 81 that cells be obtained from a well qualified cell depository 22 Other cells or cell lines may be used with. 82 the same test procedure if culture conditions are adapted to the specific needs of the cells but equivalency. 83 must be demonstrated i e appropriate responses to reference chemicals in accordance with the. 84 principles of Guidance Document No 34 21, 86 10 Cells should be checked for the absence of mycoplasma contamination by the supplier and upon.
87 arrival in the laboratory see 16 for recommendations and only used if none is found 17. 89 11 It is important that UV sensitivity of the cells is checked regularly according to the quality. 90 control procedure described in this guideline Because the UVA sensitivity of cells may increase with the. 91 number of passages BALB c 3T3 cells with a total passage number preferably less than 100 should be. OECD OCDE 432, 92 used in the assay see paragraph 29 and Annex 3. 94 Media and culture conditions, 96 12 Appropriate culture media and incubation conditions should be used for routine cell passage and. 97 during the test procedure e g for BALB c 3T3 cells these are DMEM Dulbecco s Modified Eagle s. 98 Medium supplemented with 10 new born calf serum 4 mM glutamine penicillin 100 IU and. 99 streptomycin 100 g mL and humidified incubation at 370 C 5 7 5 CO2 depending on the buffer see. 100 paragraph 17 It is important that cell culture conditions assure a cell division cycle time within the normal. 101 historical range of the cells or cell line used. 103 Preparation of cultures, 105 13 Cells from frozen stock cultures are seeded in culture medium at an appropriate density and. 106 subcultured at least once before they are used in the in vitro 3T3 NRU phototoxicity test. 108 14 Cells used for the phototoxicity test are seeded in culture medium at the appropriate density so. 109 that cultures will not reach confluence by the end of the test i e when cell viability is determined 48 h. 110 after seeding of the cells For BALB c 3T3 cells grown in 96 well plates the recommended cell seeding. 111 density is 1 x 104 cells per well, 113 15 For each test chemical cells are seeded identically in two separate 96 well plates which are then. 114 taken concurrently through the entire test procedure under identical culture conditions except for the time. 115 period where one of the plates is irradiated Irr and the other one is kept in the dark Irr. 117 Preparation of test chemical, 119 16 Test chemicals must be prepared fresh on the day of testing unless data demonstrate their stability.
120 in storage It is recommended that all chemical handling and the initial treatment of cells be performed. 121 under light conditions that would avoid photoactivation or degradation of the test chemical prior to. 122 irradiation, 124 17 Test chemicals shall be dissolved in buffered salt solutions e g Earle s or Hanks Balanced Salt. 125 Solution EBSS or HBSS or other physiologically balanced buffer solutions which must be free from. 126 protein components and light absorbing components e g pH indicator colours and vitamins to avoid. 127 interference during irradiation Since during irradiation cells are kept for about 50 minutes outside of the. 128 CO2 incubator care has to be taken to avoid alkalisation If weak buffers are used e g DPBS or EBSS. 129 this can be achieved by incubating the cells at 7 5 CO2 If the cells are incubated at 5 CO2 only a. 130 stronger buffer e g HEPES should be selected, 132 18 Test chemicals of limited solubility in water should be dissolved in an appropriate intermediate. 133 solvent If an intermediate solvent is used it must be present at a constant volume in all cultures i e in the. 134 solvent controls as well as in all concentrations of the test chemical and be non cytotoxic at that. 135 concentration, 137 19 Test chemical concentrations should be selected so as to avoid precipitation or cloudy solutions. 138 Dimethylsulphoxide DMSO and ethanol EtOH are the recommended solvents Other solvents of low. 139 cytotoxicity may be appropriate Prior to use all solvents should be assessed for specific properties e g. 432 OECD OCDE, 140 reaction with the test chemical quenching of the phototoxic effect radical scavenging properties and or. 141 chemical stability in the solvent, 143 20 Vortex mixing sonication and or warming to appropriate temperatures may be used to aid.
144 solubilisation unless this compromises the stability of the test chemical. 146 21 UV light absorption is a requirement of chemicals acting as phototoxicants and chemicals that do. 147 not have a MEC greater than 1000 L mol 1 cm 1 at any wavelength between 290 and 700 nm are not. 148 considered sufficiently photoreactive to result in phototoxicity 5 Thus UV absorption of MEC 1000 L. 149 mol 1 cm 1 between 290 and 700 nm should be considered a threshold requirement for testing phototoxicity. 152 Irradiation Conditions, 154 22 Light source i e solar stimulator The choice of an appropriate solar stimulator and filters is a. 155 crucial factor in phototoxicity testing Light of the UVA and visible regions is usually associated with. 156 phototoxic reactions in vivo 3 18 whereas generally UVB is of less relevance but is highly cytotoxic. 157 the cytotoxicity increases 1000 fold as the wavelength goes from 313 to 280 nm 19 Acceptable solar. 158 simulators emit the entire solar spectrum 290 nm through 700 nm and adjustment of the spectrum can be. 159 performed using filters to attenuate UVB while allowing UVA and visible light see Annex 3. 160 Furthermore the wavelengths doses employed and light source equipment used e g open or closed. 161 system should not be unduly deleterious to the test system e g emission of heat wavelengths in the. 162 infrared region, 164 23 Simulation of sunlight with solar simulators is considered the optimal artificial light source The. 165 irradiation power distribution of the filtered solar simulator should be close to that of outdoor daylight. 166 given in 20 Both xenon arcs and doped mercury metal halide arcs are used as solar simulators 21. 167 The latter have the advantage of emitting less heat and being cheaper but the match to sunlight is less. 168 perfect compared to that of xenon arcs All solar simulators emit significant quantities of UVB and should. 169 be suitably filtered to attenuate the highly cytotoxic UVB wavelengths Annex 1 Because cell culture. 170 plastic materials contain UV stabilisers the spectrum should be measured through the same type of 96 well. 171 plate lid as will be used in the assay Irrespective of measures taken to attenuate parts of the spectrum by. 172 filtering or by unavoidable filter effects of the equipment the spectrum recorded below these filters should. 173 not deviate from standardised outdoor daylight 20 External light standard D65 the internationally. 174 recognized emission standard for outdoor daylight is provided in ISO DIS 18909 2006 An example of the. 175 spectral irradiance distribution of the filtered solar simulator used in the validation study of the in vitro 3T3. 176 NRU phototoxicity test is given in 9 22 See also Annex 3 Figure 1. 178 24 Dosimetry The intensity of light irradiance should be regularly checked before each. 179 phototoxicity test using a suitable broadband UVA meter Annex 1 Irradiance should be measured. 180 through the same type of 96 well plate lid as will be used in the assay The UVA meter must have been. 181 calibrated to the source At greater intervals an externally calibrated reference UV vis spectroradiometer. 182 should be used to measure spectral irradiance of the filtered light source on site and to adjust the. 183 calibration of the broadband UVA meter if needed Alternatively regular calibration of the UVA meter. 184 could be performance at a central calibration laboratory provided that this facility is equipped with an. 185 identical light source filter combination, 187 25 A dose of 5 J cm2 as measured in the UVA range was determined to be non cytotoxic to. OECD OCDE 432, 188 BALB c 3T3 cells and sufficiently potent to excite chemicals to elicit phototoxic reactions 7 23 To. 189 achieve 5 J cm2 within a time period of 50 min irradiance was adjusted to 1 7 mW cm2 See Annex 3. 190 Figure 2 Alternate exposure times and or irradiance values may be used to achieve 5 J cm2 using the. 191 formula, irradiatio n dose J cm 2 1000, 193 t min 1 J 1 Wsec.
irradiance mW cm 2 60, 195 26 Similarly if another cell line or a different light source is used the irradiation should be. 196 calibrated so that a dose regimen can be selected that is not deleterious to the cells but sufficient to excite. 197 standard phototoxins e g proficiency chemicals described in Table 1 27. 199 Test conditions, 201 Test chemical concentrations. 203 27 The ranges of concentrations of a chemical tested in the presence Irr and in the absence. 204 Irr of light should be adequately determined in dose range finding experiments It may be useful to. 205 assess solubility initially and at 60 min or whatever treatment time is to be used as solubility can change. 206 during time or during the course of exposure To avoid toxicity induced by improper culture conditions or. 207 by highly acidic or alkaline chemicals the pH of the cell cultures with added test chemical should be in the. 208 range 6 to 8, 210 28 The highest concentration of the test chemical should be within physiological test conditions. 211 e g osmotic and pH stress should be avoided Depending on the test chemical it may be necessary to. 212 consider other physico chemical properties as factors limiting the highest test concentration For relatively. 213 insoluble chemicals that are not toxic at concentrations up to the saturation point the highest achievable. 214 concentration should be tested For non cytotoxic chemicals no IC50 value up to precipitation it might be. 215 useful to demonstrate the solubility limit under assay conditions In this case including two or three. 216 concentrations in the main experiment that will likely show precipitation may be useful The maximum. 217 concentration of a test chemical should not exceed 1000 g mL osmolality should not exceed 10 mM In. 218 many cases the maximum concentration can be reduced to 100 g mL since compounds without any. 219 significant cytotoxicity under irradiation up to this limit can be considered as being devoid of relevant. 220 phototoxicity 5 Higher maximum concentration without irradiation might still be considered to establish. 221 IC50 values for PIF calculation A geometric dilution series of 8 test concentrations with a constant dilution. 222 factor should be used see paragraph 47, 224 29 If there is information from a range finding experiment that the test chemical is not cytotoxic up. 225 to the limit concentration in the dark experiment Irr but is highly cytotoxic when irradiated Irr the. 226 concentration ranges to be selected for the Irr experiment may differ from those selected for the Irr. 125 Solution EBSS or HBSS or other physiologically balanced buffer solutions which must be free from 126 protein components and light absorbing components e g pH indicator colours and vitamins to avoid 127 interference during irradiation Since during irradiation cells are kept for about 50 minutes outside of the 128 CO 2

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