OECD/OCDE
护理研究生考试科目
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Adopted: 2 October 2012
© OECD, (2012)
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OECD GUIDELINE FOR THE TESTING OF CHEMICALS Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants
INTRODUCTION
1. The Fluorescein Leakage (FL) test method is an in vitro test method that can be ud under cert
ain circumstances and with specific limitations to classify chemicals (substances and mixtures) as ocular corrosives and vere irritants, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Category 1), the European Union (EU) Regulation on Classification, Labelling and Packaging of Substances and Mixtures (CLP) (Category 1), and the U.S. Environmental Protection Agency (EPA) (Category I) (1) (2) (3). For the purpo of this Test Guideline, vere irritants are defined as chemicals that cau tissue damage in the eye following test substance administration that is not reversible within 21 days or caus rious physical decay of vision, while ocular corrosives are chemicals that cau irreversible tissue damage to the eye. The chemicals are classified as UN GHS Category 1, EU CLP Category 1, or U.S. EPA Category I.
2. While the FL test method is not considered valid as a complete replacement for the in vivo rabbit eye test, the FL is recommended for u as part of a tiered testing strategy for regulatory classification and labelling. Thus, the FL is recommended as an initial step within a Top-Down approach to identify ocular corrosives/vere irritants, specifically for limited types of chemicals (i.e. water soluble substances and mixtures) (4)(5).
3. It is currently generally accepted that, in the foreeable future, no single in vitro eye irritation test
will be able to replace the in vivo eye test (TG 405 (6)) to predict across the full range of irritation for different chemical class. However, strategic combinations of veral alternative test methods within a (tiered) testing strategy may be able to replace the in vivo eye test (5). The Top-Down approach (5) is designed to be ud when, bad on existing information, a chemical is expected to have high irritancy potential.
Bad on the prediction model detailed in paragraph 35, the FL test method can identify Category 1; EU CLP Category 1; U.S. EPA Category I) without any further testing. The same is assumed for mixtures although mixtures were not ud in the validation. Therefore, the FL test method may be ud to determine the eye irritancy/corrosivity of chemicals, following the
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quential testing strategy of TG 405 (6). However, a chemical that is not predicted as ocular corrosive or vere irritant with the FL test method would need to be tested in one or more additional test methods (in vitro and/or in vivo) that are capable of accurately identifying i) chemicals that are in vitro fal negative ocular corrosives/vere irritants in the FL (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I); ii) chemicals that are not classified for eye corrosion/irritation (UN
GHS No Category; EU CLP No Category; U.S. EPA Category IV); and/or iii) chemicals that are moderate/mild eye irritants (UN GHS Categories 2A and 2B; EU CLP Category 2; U.S. EPA Categories II and III).
5. The purpo of this Test Guideline is to describe the procedures ud to evaluate the potential ocular corrosivity or vere irritancy of a test substance as measured by its ability to induce damage to an impermeable confluent epithelial monolayer. The integrity of trans-epithelial permeability is a major function of an epithelium such as that found in the conjunctiva and the cornea. Trans-epithelial permeability is controlled by various tight junctions. Increasing the permeability of the corneal epithelium in vivo has been shown to correlate with the level of inflammation and surface damage obrved as eye irritation develops.
6. In the FL test method, toxic effects after a short exposure time to the test substance are measured by an increa in permeability of sodium fluorescein through the epithelial monolayer of Madin-Darby Canine Kidney (MDCK) cells cultured on permeable inrts. The amount of fluorescein leakage that occurs is proportional to the chemical-induced damage to the tight junctions, desmosomal junctions and cell membranes, and can be ud to estimate the ocular toxicity potential of a test substance. Annex I provides a diagram of MDCK cells grown on an inrt membrane for the
FL test method.
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7. Definitions are provided in Annex II.
INITIAL CONSIDERATIONS AND LIMITATIONS
8. This Test Guideline is bad on the INVITTOX protocol No. 71 (7) that has been evaluated in an international validation study by the European Centre for the Validation of Alternative Methods (ECVAM) (8), in collaboration with the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the Japane Center for the Validation of Alternative Methods (JaCVAM).
9. The FL test method is not recommended for the identification of chemicals which should be classified as mild/moderate irritants or of chemicals which should not be classified for ocular irritation (substances and mixtures) (i.e. GHS Cat. 2A/2B, no category; EU CLP Cat. 2, no category; US EPA Cat. II/III/IV), as demonstrated by the validation study (4) (8).
10. The test method is only applicable to water soluble chemicals (substances and mixtures). The ocular vere irritation potential of chemicals that are water soluble and/or where the toxic effect is n
ot affected by dilution is generally predicted accurately using the FL test method (8). To categori a chemical as water soluble, under experimental conditions, it should be soluble in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, Hanks’ Buffered Salt Solution (HBSS) at a concentration ≥ 250 mg/mL (one do above the cut-off of 100 mg/mL). However, if the test substance is soluble below the concentration 100 mg/mL,
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OECD/OCDE 460 but already induces a FL induction of 20 % at that concentration (meaning FL20 < 100 mg/mL), it can still be classified as GHS Cat. 1 or EPA Cat. 1.
11. The identified limitations for this test method exclude strong acids and bas, cell fixatives and highly volatile chemicals from the applicability domain. The chemicals have mechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponification or specific reactive chemistries. Other identified limitations for this method are bad upon the results for the predictive capacity for coloured and viscous test substance (8). It is suggested that both types of chemicals are difficult to remove from the monolayer following the short exposure period and that predictivity of the
test method could be improved if a higher number of washing steps was ud. Solid chemicals suspended in liquid have the propensity to precipitate out and the final concentration to cells can be difficult to determine. When substances within the chemical and physical class are excluded from the databa, the accuracy of FL across the EU, EPA, and GHS classification systems is substantially improved (8).
12. Bad on the purpo of this test method (i.e. to identify ocular corrosives/vere irritants only), fal negative rates (e Paragraph 13) are not critical since such substances would be subquently tested with other adequately validated in vitro tests or in rabbits, depending on regulatory requirements, using a quential testing strategy in a weight of evidence approach (6) (e also paragraphs 3 and 4).开机时间
13. Other identified limitations of the FL test method are bad on fal negative and fal positive rates. When ud as an initial step within a Top-Down approach to identify water soluble ocular corrosive/vere irritant substances and mixtures (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I), the fal positive rate for the FL test method ranged from 7% (7/103; UN GHS and EU CLP) to 9% (9/99; U.S. EPA) and the fal negative rate ranged from 54% (15/28; U.S. EPA) to 56% (27/48; UN GHS and EU CLP) when compared to in vivo results. Chemical groups showing fals
e positive and/or fal negative results in the FL test method are not defined here.
14. Certain technical limitations are specific to the MDCK cell culture. The tight junctions that block the passage of the sodium-fluorescein dye through the monolayer are increasingly compromid with increasing cell passage number. Incomplete formation of the tight junctions results in incread FL in the non-treated control. Therefore, a defined permissible maximal leakage in the non-treated controls is important (e paragraph 38: 0% leakage). As with all in vitro assays there is the potential for the cells to become transformed over time, thus it is vital that passage number ranges for the assays are stated.
15. The current applicability domain might be incread in some cas, but only after analyzing an expanded data t of studied test substances, preferably acquired through testing (4). This Test Guideline will be updated accordingly as new information and data are considered.
hotel booking16. For any laboratory initially establishing this assay, the proficiency chemicals provided in Annex III should be ud. Laboratories can u the chemicals to demonstrate their technical competence in performing the FL test method prior to submitting FL assay data for regulatory hazard classification purpos.
gliderPRINCIPLE OF THE TEST
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17. The FL test method is a cytotoxicity and cell-function bad in vitro assay that is performed on a confluent monolayer of MDCK CB997 tubular epithelial cells that are grown on mi-permeable inrts and model the non-proliferating state of the in vivo corneal epithelium. The MDCK cell line is well established and forms tight junctions and desmosomal junctions similar to tho found on the apical side of conjunctival and corneal epithelia. Tight and desmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal epithelium. Loss of trans-epithelial impermeability, due to damaged tight junctions and desmosomal junctions, is one of the early events in chemical-induced ocular irritation.
18. The test substance is applied to the confluent layer of cells grown on the apical side of the inrt. A short 1 min exposure is routinely ud to reflect the normal clearance rate in human exposures. An
advantage of the short exposure period is that water-bad substances and mixtures can be tested neat, if they can be easily removed after the exposure period. This allows more direct comparisons of the results with the chemical effects in humans. The test substance is then removed and the non-toxic, highly fluorescent sodium-fluorescein dye is added to the apical side of the monolayer for 30 minutes. The damage caud by the test substance to the tight junctions is determined by the amount of fluorescein which leaks through the cell layer within a defined period of time.
19. The amount of sodium-fluorescein dye that pass through the monolayer and the inrt membrane into a t volume of solution prent in the well (to which the sodium-fluorescein dye leaks in) is determined by measuring spectrofluorometrically the fluorescein concentration in the well. The amount of fluorescein leakage (FL) is calculated with reference to fluorence intensity (FI) readings from two controls: a blank control, and a maximum leakage control. The percentage of leakage and therefore amount of damage to the tight junctions is expresd, relative to the controls, for each of the t concentrations of the test substance. Then the FL20 (i.e. concentration that caus 20% FL relative to the value recorded for the untreated confluent monolayer and inrts without cells), is calculated. The FL20 (mg/mL) value is ud in the prediction model for identification of ocular corrosives and vere irritants (e paragraph 35).
20. Recovery is an important part of a test substance’s toxicity profile that is also assd by the in vivo ocular irritation test. Preliminary analys indicated that recovery data (up to 72 h following the chemical exposure) could potentially increa the predictive capacity of INVITTOX Protocol 71 but further evaluation is needed and would benefit from additional data, preferably acquired by further testing (7). This Test Guideline will be updated accordingly as new information and data are considered.
PROCEDURE
Preparation of the cellular monolayer雅思口语考试评分标准
frastructure21. The monolayer of MDCK CB997 cells is prepared using sub-confluent cells growing in cell culture flasks in DMEM/Nutrient Mix F12 (1x concentrate with L-glutamine, 15 mM HEPES, calcium (at a concentration of 1.0-1.8 mM) and 10% heat-inactivated FCS/FBS). Importantly, all media/solutions ud throughout the FL assay should contain calcium at a concentration between 1.8 mM (200 mg/L) and 1.0 mM (111 mg/L) to ensure tight junction formation and integrity. Cell passage number range should be controlled to ensure even and
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OECD/OCDE 460 reproducible tight junctions formation. Preferably, the cells should be within the passage range 3-30 from thawing becau cells within this passage range have similar functionality, which aids assay results to be reproducible.
22. Prior to performing the FL test method, the cells are detached from the flask by trypsinisation, centrifuged and an appropriate amount of cells is eded into the inrts placed in 24-well plates (e Annex I). Twelve mm diameter inrts with membrane of mixed cellulo esters, a thickness of 80-150 µm and a pore size of 0.45 µm, should be ud to ed the cells. In the validation study, Millicell-HA 12 mm inrts were ud. The properties of the inrt and membrane type are important as the may affect cell growth and chemical binding. Certain types of chemicals may bind to the Millicell-HA inrt membrane, which could affect the interpretation of results. Proficiency chemicals (e Annex III) should be ud to demonstrate equivalency if other membranes are ud.
23. Chemical binding to the inrt membrane is more common for cationic chemicals, such as benzalkonium chloride, which are attracted to the positively charged membrane (8). Chemical binding to the inrt membrane may increa the chemical exposure period, leading to an over-esti
cnn提问英国39死事件mation of the toxic potential of the chemical, but can also physically reduce the leakage of fluorescein through the inrt by binding of the dye to the cationic chemical bound to the inrt membrane, leading to an under-estimation of the toxic potential of the chemical. This can be readily monitored by exposing the membrane alone to the top concentration of the chemical tested and then adding sodium-fluorescein dye at the normal concentration for the standard time (no cell control). If binding of the sodium-fluorescein dye occurs, the inrt membrane appears yellow after the test material has been washed-off. Thus, it is esntial to know the binding properties of the test substance in order to be able to interpret the effect of the chemical on the cells.
24. Cell eding on inrts should produce a confluent monolayer at the time of chemical exposure. 1.6 x 105 cells should be added per inrt (400 µL of a cell suspension with a density of 4 x 105 cells / mL). Under the conditions, a confluent monolayer is usually obtained after 96 hours in culture. Inrts should be examined visually prior to eding, so as to ensure that any damages recorded at the visual control described at paragraph 30 is due to handling.
25. The MDCK cell cultures should be kept in incubators in a humidified atmosphere, at 5% ± 1% CO2and 37 ± 1 ºC. The cells should be free of contamination by bacteria, virus, mycoplasma and fungi.
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Application of the Test and Control Chemicals
26. A fresh stock solution of test substance should be prepared for each experimental run and ud within 30 minutes of preparation. Test substances should be prepared in calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS to avoid rum protein binding. Solubility of the chemical at 250 mg/mL in HBSS should be assd prior to testing. If at this concentration the chemical forms a stable suspension or emulsion (i.e.maintains uniformity and does not ttle or parate into more than one pha) over 30 minutes, HBSS can still be ud as solvent. However, if the chemical is found to be insoluble in HBSS at this concentration, the u of other test methods instead of FL should be considered. The u of light mineral oil as a solvent, in cas where the chemical is found to be insoluble in HBSS, should be
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considered with caution as there is not enough data available to conclude on the performance of the FL assay under such conditions.
27. All chemicals to be tested are prepared in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS from the stock solution, at five fixed concentrations diluted on a weight per volume basis: 1, 25, 100, 250 mg/mL and a neat or a saturated solution. When testing a solid chemical, a very high concentration of 750 mg/mL should be included. This concentration of chemical may have to be applied on the cells using a positive displacement pipette. If the toxicity is found to be between 25 and 100 mg/mL, the following additional concentrations should be tested twice: 1, 25, 50, 75, 100 mg/mL. The FL20value should be derived from the concentrations provided the acceptance criteria were met.
28. The test substances are applied to the confluent cell monolayers after removal of the cell culture medium and washing twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS. Previously, the filters have been visually checked for any pre-existing damages that could be fally attributed to potential incompatibilities with test chemicals. At least three replicates should be ud for each concentration of the test substance and for the controls in each run. After 1 min of exposure at room temperature, the test substance should be carefully removed by aspiration, the monolayer should be washed twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS, and the fluorescein le
akage should be immediately measured. 29. Concurrent negative (NC) and positive controls (PC) should be ud in each run to demonstrate that monolayer integrity (NC) and nsitivity of the cells (PC) are within a defined historical acceptance range. The suggested PC chemical is Brij 35 (CAS No. 9002-92-0) at 100 mg/mL. This concentration should give approximately 30% fluorescein leakage (acceptable range 20-40% fluorescein leakage, i.e. damage to cell layer). The suggested NC chemical is calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS (untreated, blank control).
A maximum leakage control should also be included in each run to allow for the calculation of FL20 values. Maximum leakage is determined using a control inrt without cells.
Determination of fluorescein permeability
30. Immediately after removal of the test and control substances, 400μL of 0.1 mg/mL sodium-fluorescein solution (0.01% (w/v) in calcium-containing [at a concentration of 1.0-1.8 mM], phenol red-free, HBSS) is added to the Millicell-HA inrts. The cultures are kept for 30 minutes at room temperature. At the end of the incubation with fluorescein, the inrts are carefully removed from each well. Visual check is performed on each filter and any damage which may have occurred during handling is recorded.
31. The amount of fluorescein that leaked through the monolayer and the inrt is quantified in the solution which remained in the wells after removal of the inrts. Measurements are done in a spectrofluorometer at excitation and emission wavelengths of 485 nm and 530 nm, respectively. The nsitivity of the spectrofluorometer should be t so that there is the highest numerical difference between the maximum FL (inrt with no cells) and the minimum FL (inrt with confluent monolayer treated with NC). Becau of the differences in the ud spectrofluorometer, it is suggested that a nsitivity is ud which will give fluorescence intensity > 4000 at the maximum fluorescein leakage control. The maximum FL value should not be
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