natgillin/translations-raw huggingface dataset:
In the FL test method, toxic effects after a short exposure time to the test chemical are measured by an increase in permeability of sodium fluorescein through the epithelial monolayer of Madin-Darby Canine Kidney (MDCK) cells cultured on permeable inserts.
Preskusna metoda FL se priporoča samo za opredeljevanje vodotopnih jedkih in zelo dražilnih snovi za oči(kategorija 1 po GHS ZN, kategorija 1 po CLP EU, kategorija I po US EPA) (glej odstavka 1 in 10).
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 used to estimate the ocular toxicity potential of a test chemical.
Za opredelitev vodotopnih kemikalij (snovi in zmesi) (3) (6) (7) kot „povzroča hudo poškodbo oči“ (kategorija 1 po GHS ZN/CLP EU) ali „jedka ali zelo dražilna snov za oči“ (kategorija I po US EPA) bi morala preskusna kemikalija pri koncentraciji ≤ 100 mg/ml inducirati vrednost FL20.
Appendix 1 provides a diagram of MDCK cells grown on an insert membrane for the FL test method.
Sprejemljivost rezultatov
Definitions are provided in Appendix 2.
Srednja vrednost pri največjem uhajanju fluoresceina (x) mora biti višja od 4000 (glej odstavek 31), srednja vrednost pri 0-odstotnem uhajanju (y) mora biti enaka ali nižja od 300, srednja vrednost pri 100-odstotnem uhajanju (z) pa mora biti med 3700 in 6000.
This test method is based on the INVITTOX protocol No. 71 (6) that has been evaluated in an international validation study by the European Centre for the Validation of Alternative Methods (ECVAM), in collaboration with the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM).
Preskus se obravnava kot sprejemljiv, če je bila pri pozitivni kontroli opažena 20- do 40-odstotna poškodovanost celične plasti (meritev kot % uhajanja fluoresceina).
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.
Podatki
2A/2B, no category; EU CLP Cat.
Podatki iz posameznih jamic s ponovljenimi vzorci (npr. vrednosti intenzitete fluorescence in izračunani podatki o odstotku FL za vsako preskusno kemikalijo, vključno z razvrstitvijo) za vsako ponovitev preskusa morajo biti navedeni v obliki preglednice.
2, no category; US EPA Cat.
Poleg tega je treba v poročilu navesti srednje vrednosti ± standardni odklon za posamezne ponovljene meritve v vsaki ponovitvi preskusa.
II/III/IV), as demonstrated by the validation study (3) (7).
številka CAS kemikalije, če je znana;
The test method is only applicable to water soluble chemicals (substances and mixtures).
čistost in sestava snovi ali zmesi (v masnih deležih), če so te informacije na voljo;
The ocular severe irritation potential of chemicals that are water soluble and/or where the toxic effect is not affected by dilution is generally predicted accurately using the FL test method (7).
fizikalno-kemijske lastnosti, pomembne za izvedbo študije (npr. agregatno stanje, hlapnost, vrednost pH, stabilnost, topnost v vodi, kemijski razred);
To categorise 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' Balanced Salt Solution (HBSS) at a concentration ≥ 250 mg/ml (one dose above the cut-off of 100 mg/ml).
obdelava preskusne/kontrolne kemikalije pred preskušanjem, če je ustrezno (npr. segrevanje, mletje);
However, if the test chemical is soluble below the concentration 100 mg/ml, but already induces a FL induction of 20 % at that concentration (meaning FL20 < 100 mg/ml), it can still be classified as GHS Cat.
pogoji shranjevanja.
1 or EPA Cat.
Utemeljitev preskusne metode in uporabljenega protokola:
I.
vključevati mora preudarke v zvezi s področjem uporabe in omejitve preskusne metode.
The identified limitations for this test method exclude strong acids and bases, cell fixatives and highly volatile chemicals from the applicability domain.
opis uporabljenega celičnega sistema, vključno s potrdilom o avtentičnosti in statusom mikoplazme celične linije;
These chemicals have mechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponification or specific reactive chemistries.
podrobnosti o uporabljenem preskusnem postopku;
Other identified limitations for this method are based upon the results for the predictive capacity for coloured and viscous test chemical (7).
uporabljene koncentracije kemikalije;
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 used.
trajanje izpostavljenosti preskusni kemikaliji;
Solid chemicals suspended in liquid have the propensity to precipitate out and the final concentration to cells can be difficult to determine.
trajanje inkubacije s fluoresceinom;
When chemicals within these chemical and physical classes are excluded from the database, the accuracy of FL across the EU, EPA, and GHS classification systems is substantially improved (7).
opis uporabljenih meril za ocenjevanje;
Based on the purpose of this test method (i.e. to identify ocular corrosives/severe irritants only), false negative rates (see Paragraph 13) are not critical since such chemicals would be subsequently tested with other adequately validated in vitro tests or in rabbits, depending on regulatory requirements, using a sequential testing strategy in a weight of evidence approach (5) (see also paragraphs 3 and 4).
navedba podatkov o modelu iz preteklih preskusov (npr. negativne in pozitivne kontrole, primerjalne kemikalije, če je ustrezno);
Other identified limitations of the FL test method are based on false negative and false positive rates.
informacije o tehnični usposobljenosti, ki jo je dokazal laboratorij.
When used as an initial step within a Top-Down approach to identify water soluble ocular corrosive/severe irritant substances and mixtures (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I), the false 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 false 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.
preglednice s podatki o posameznih preskusnih kemikalijah, kontrolah za vsako ponovitev preskusa in vsako ponovljeno meritev (vključno s posameznimi rezultati, srednjimi vrednostmi in standardnimi odkloni);
Chemical groups showing false positive and/or false negative results in the FL test method are not defined here.
izpeljane razvrstitve z navedbo napovednega modela in/ali uporabljenih meril za odločitev;
Certain technical limitations are specific to the MDCK cell culture.
vključevati mora preudarke o nedokončnem rezultatu (odstavek 35: FL20 > 100 mg/ml) in nadaljnjem preskušanju.
The tight junctions that block the passage of the sodium-fluorescein dye through the monolayer are increasingly compromised with increasing cell passage number.
Sklepne ugotovitve
Incomplete formation of the tight junctions results in increased FL in the non-treated control.
Na voljo na: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html].
Therefore, a defined permissible maximal leakage in the non-treated controls is important (see paragraph 38: 0 % leakage).
Poglavje B.5 te priloge, Akutno draženje oči/jedkost za oči.
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.
Na voljo na: [http://ecvam-dbalm.jrc.ec.europa.eu.
The current applicability domain might be increased in some cases, but only after analysing an expanded data set of studied test chemicals, preferably acquired through testing (3).
OECD (2005).
This test method will be updated accordingly as new information and data are considered.
Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment št. 34.
For any laboratory initially establishing this assay, the proficiency chemicals provided in Appendix 3 should be used.
DIAGRAM CELIC MDCK, KI SE GOJIJO NA MEMBRANI VSTAVKA ZA PRESKUSNO METODO FL
Laboratories can use these chemicals to demonstrate their technical competence in performing the FL test method prior to submitting FL assay data for regulatory hazard classification purposes.
Konfluentni sloj celic MDCK se goji na polprepustni membrani vstavka.
The FL test method is a cytotoxicity and cell-function based in vitro assay that is performed on a confluent monolayer of MDCK CB997 tubular epithelial cells that are grown on semi-permeable inserts and model the non-proliferating state of the in vivo corneal epithelium.
Vstavki se položijo v jamice plošče s 24 jamicami.
The MDCK cell line is well established and forms tight junctions and desmosomal junctions similar to those found on the apical side of conjunctival and corneal epithelia.
Vir slike: Wilkinson, P. J. (2006).
Tight and desmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal epithelium.
Kategorija I po EPA
Loss of trans-epithelial impermeability, due to damaged tight junctions and desmosomal junctions, is one of the early events in chemical-induced ocular irritation.
kemikalije, ki so jedke (nepopravljivo uničenje očesnega tkiva) ali vplivajo na roženico oziroma jo dražijo več kot 21 dni (2).
The test chemical is applied to the confluent layer of cells grown on the apical side of the insert.
CLP EU (Uredba (ES) št. 1272/2008 o razvrščanju, označevanju in pakiranju snovi ter zmesi)
A short 1 min exposure is routinely used to reflect the normal clearance rate in human exposures.
v Evropski uniji (EU) izvaja Globalno usklajeni sistem za razvrščanje kemikalij (snovi in zmesi) Združenih narodov (GHS ZN).
An advantage of the short exposure period is that water-based substances and mixtures can be tested neat, if they can be easily removed after the exposure period.
to vrednost je mogoče oceniti z opredelitvijo koncentracije, pri kateri preskušene kemikalije povzročijo 20-odstotno uhajanje fluoresceina skozi celično plast.
This allows more direct comparisons of the results with the chemical effects in humans.
Uhajanje fluoresceina
The test chemical is then removed and the non-toxic, highly fluorescent sodium-fluorescein dye is added to the apical side of the monolayer for 30 minutes.
količina fluoresceina, ki prodre skozi celično plast; izmeri se s spektrofluorimetrom.
The damage caused by the test chemical to the tight junctions is determined by the amount of fluorescein which leaks through the cell layer within a defined period of time.
GHS (Globalno usklajeni sistem Združenih narodov (ZN) za razvrščanje in označevanje kemikalij)
The amount of sodium-fluorescein dye that passes through the monolayer and the insert membrane into a set volume of solution present in the well (to which the sodium-fluorescein dye leaks in) is determined by measuring spectrofluorometrically the fluorescein concentration in the well.
sistem za razvrščanje kemikalij (snovi in zmesi) v skladu s standardiziranimi vrstami in stopnjami fizičnih, zdravstvenih in okoljskih nevarnosti ter za obravnavanje ustreznega označevanja, na primer s piktogrami, opozorilnimi besedami, stavki o nevarnosti, previdnostnimi stavki in varnostnimi listi, da bi se razširile informacije o škodljivih učinkih kemikalij ter s tem zaščitili ljudje (vključno z delodajalci, delavci, prevozniki, potrošniki in reševalci) in okolje.
The amount of fluorescein leakage (FL) is calculated with reference to fluoresence intensity (FI) readings from two controls: a blank control, and a maximum leakage control.
Kategorija 1 po GHS
The percentage of leakage and therefore amount of damage to the tight junctions is expressed, relative to these controls, for each of the set concentrations of the test chemical.
v okviru GHS ZN se uporablja kot zmes ali raztopina iz dveh ali več snovi, ki v njej ne reagirajo.
Then the FL20 (i.e. concentration that causes 20 % FL relative to the value recorded for the untreated confluent monolayer and inserts without cells), is calculated.
Brez razvrstitve
The FL20 (mg/ml) value is used in the prediction model for identification of ocular corrosives and severe irritants (see paragraph 35).
kemikalije,ki niso razvrščene med dražilne snovi za oči kategorije 1, 2A ali 2B po GHS ZN; kategorije 1 ali 2 po CLP EU; ali kategorije I, II ali III po US EPA.
Preparation of the cellular monolayer
kemikalija, ki po dodajanju na sprednjo površino očesa povzroča popravljivo poškodbo očesa; (b) kemikalije, ki so razvrščene med dražilne snovi za oči kategorije 2A ali 2B po GHS ZN; kategorije 2 po CLP EU; ali kategorije II ali III po US EPA.
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).
Zelo dražilna snov za oči
Importantly, all media/solutions used 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.
kemikalija, ki po dodajanju na sprednjo površino očesa povzroči poškodbo očesnega tkiva, ki v 21 dneh po nanosu ne izgine, ali povzroči resno fizično okvaro vida; (b) kemikalije, ki so razvrščene med dražilne snovi za oči kategorije 1 po GHS ZN; kategorije 1 po CLP EU; ali kategorije I po US EPA.
Cell passage number range should be controlled to ensure even and reproducible tight junctions formation.
Pozitivna kontrola
Preferably, the cells should be within the passage range 3-30 from thawing because cells within this passage range have similar functionality, which aids assay results to be reproducible.
ponovljen vzorec, ki vsebuje vse sestavine preskusnega sistema in se tretira s kemikalijo, za katero je znano, da povzroči pozitiven odziv.
Prior to performing the FL test method, the cells are detached from the flask by trypsinisation, centrifuged and an appropriate amount of cells is seeded into the inserts placed in 24-well plates (see Appendix 1).
Za zagotovitev, da se lahko oceni variabilnost odziva pozitivne kontrole v daljšem časovnem obdobju, stopnja pozitivnega odziva ne sme biti prevelika.
Twelve mm diameter inserts with membrane of mixed cellulose esters, a thickness of 80-150 μm and a pore size of 0,45 μm, should be used to seed the cells.
Kemikalije za preverjanje usposobljenosti
In the validation study, Millicell-HA 12 mm inserts were used.
podskupina seznama referenčnih kemikalij, ki jih lahko uporabi laboratorij, da dokaže usposobljenost za validirano, referenčno preskusno metodo.
The properties of the insert and membrane type are important as these may affect cell growth and chemical binding.
Ustreznost upošteva tudi točnost (skladnost) preskusne metode (8).
Certain types of chemicals may bind to the Millicell-HA insert membrane, which could affect the interpretation of results.
Nadomestni preskus
Proficiency chemicals (see Appendix 3) should be used to demonstrate equivalency if other membranes are used.
preskus, ki nadomesti preskus, ki se redno uporablja in je uveljavljen za ugotavljanje nevarnosti in/ali oceno tveganja, ter za katerega je bilo ugotovljeno, da v primerjavi z uveljavljenim preskusom v vseh mogočih okoliščinah preskušanja in za vse mogoče preskusne kemikalije zagotavlja enakovredno ali izboljšano varstvo zdravja ljudi ali živali ali okolja, kot se uporablja.
Chemical binding to the insert membrane is more common for cationic chemicals, such as benzalkonium chloride, which are attracted to the charged membrane (7).
delež vseh pozitivnih/aktivnih kemikalij, ki se s preskusno metodo pravilno razvrstijo.
Chemical binding to the insert membrane may increase the chemical exposure period, leading to an over-estimation of the toxic potential of the chemical, but can also physically reduce the leakage of fluorescein through the insert by binding of the dye to the cationic chemical bound to the insert membrane, leading to an under-estimation of the toxic potential of the chemical.
Je merilo točnosti za preskusno metodo, s katero se pridobijo kategorični rezultati, in pomemben dejavnik pri ocenjevanju ustreznosti preskusne metode (8).
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).
povzročitev poškodbe očesnega tkiva ali resne fizične okvare vida po nanosu preskusne kemikalije na sprednjo površino očesa, ki ni v celoti popravljiva v 21 dneh po nanosu.
If binding of the sodium-fluorescein dye occurs, the insert membrane appears yellow after the test material has been washed-off.
netretiran vzorec, ki vsebuje vse komponente preskusnega sistema, vključno s topilom ali vehiklom, ki se obdela s kontrolnimi vzorci, tretiranimi s preskusno kemikalijo, in drugimi kontrolnimi vzorci, da se določi izhodiščni odziv za vzorce, tretirane s preskusno kemikalijo, raztopljeno v istem topilu ali vehiklu.
Thus, it is essential to know the binding properties of the test chemical in order to be able to interpret the effect of the chemical on the cells.
delež vseh negativnih/neaktivnih kemikalij, ki se s tem preskusom pravilno razvrstijo.
Cell seeding on inserts should produce a confluent monolayer at the time of chemical exposure.
Je merilo točnosti za preskusno metodo, s katero se pridobijo kategorični rezultati, in pomemben dejavnik pri ocenjevanju ustreznosti preskusne metode.
1,6 × 105 cells should be added per insert (400 μl of a cell suspension with a density of 4 × 105 cells / ml).
v okviru GHS ZN se uporablja kot kemijski element in njegove spojine, ki so v naravnem stanju ali pridobljeni s katerim koli proizvodnim postopkom, vključno z vsemi dodatki, potrebnimi za ohranitev stabilnosti produkta, in kakršnimi koli nečistotami, ki so nastale v uporabljenem postopku, vendar brez kakršnega koli topila, ki ga je mogoče ločiti, ne da bi to vplivalo na stabilnost snovi ali spremenilo njeno sestavo.
Under these conditions, a confluent monolayer is usually obtained after 96 hours in culture.
Opozoriti je treba, da točnost in zanesljivost validirane preskusne metode nista nujno zadostni, da bi bila sprejemljiva za predlagani namen (8).
Inserts should be examined visually prior to seeding, so as to ensure that any damages recorded at the visual control described at paragraph 30 is due to handling.
KEMIKALIJE ZA PREVERJANJE USPOSOBLJENOSTI ZA PRESKUSNO METODO FL
The MDCK cell cultures should be kept in incubators in a humidified atmosphere, at 5 % ± 1 % CO2 and 37 ± 1 °C.
Pred redno uporabo te preskusne metode morajo laboratoriji dokazati tehnično usposobljenost tako, da pravilno opredelijo razvrstitev med jedke snovi za oči za 8 kemikalij, ki so priporočene v preglednici 1.
The cells should be free of contamination by bacteria, viruses, mycoplasma and fungi.
Te kemikalije so bile izbrane tako, da predstavljajo vrsto odzivov za lokalno draženje/jedkost za oči, ki temelji na rezultatih očesne študije in vivo na kuncih (TG 405, PM B.5(5)) (tj. kategorije 1, 2A in 2B ali brez razvrstitve po GHS ZN).
Application of the Test and Control Chemicals
Ob upoštevanju validirane uporabnosti preskusa FL (tj. opredeljevanje samo jedkih/zelo dražilnih snovi za oči) pa za dokaz usposobljenosti obstajata samo dva rezultata preskusa za namene razvrstitve (jedka/zelo dražilna snov za oči ali nejedka/lažje dražilna snov za oči).
A fresh stock solution of test chemical should be prepared for each experimental run and used within 30 minutes of preparation.
Druga merila za izbiro so bila dostopnost kemikalij na trgu, razpoložljivost visokokakovostnih referenčnih podatkov in vivo in visokokakovostni podatki iz preskusne metode FL.
Test chemicals should be prepared in calcium-containing (at a concentration of 1,0-1,8 mM), phenol red-free, HBSS to avoid serum protein binding.
Zato so bile kemikalije za preverjanje usposobljenosti izbrane na podlagi dokumenta „Fluorescein Leakage Assay Background Review Document as an Alternative Method for Eye Irritation Testing“ (dokument o pregledu ozadja v preskusu uhajanja fluoresceina kot alternativne metode za preskušanje dražilnosti oči) (8), ki je bil uporabljen za retrospektivno validacijo preskusne metode FL.
Solubility of the chemical at 250 mg/ml in HBSS should be assessed prior to testing.
Priporočene kemikalije za dokazovanje tehnične usposobljenosti za FL
If at this concentration the chemical forms a stable suspension or emulsion (i.e. maintains uniformity and does not settle or separate into more than one phase) over 30 minutes, HBSS can still be used as solvent.
Kratice: št. CAS = registrska številka Službe za izmenjavo kemijskih izvlečkov
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.
prometazin hidroklorid
When testing a solid chemical, a very high concentration of 750 mg/ml should be included.
amin/amidin, heterociklična spojina, organska žveplova spojina
This concentration of chemical may have to be applied on the cells using a positive displacement pipette.
natrijev hidroksid (10 %)
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.
baza
The FL20 value should be derived from these concentrations provided the acceptance criteria were met.
natrijev lavril sulfat (15 %)
The test chemicals are applied to the confluent cell monolayers after removal of the cell culture medium and washing twice with sterile, warm (37 °C), calcium-containing (at a concentration of 1,0-1,8 mM), phenol red-free, HBSS.
karboksilna kislina (sol)
Previously, the filters have been visually checked for any pre-existing damages that could be falsely attributed to potential incompatibilities with test chemicals.
arboksi-benzaldehid
At least three replicates should be used for each concentration of the test chemical and for the controls in each run.
karboksilna kislina, aldehid
After 1 min of exposure at room temperature, the test chemical should be carefully removed by aspiration, the monolayer should be washed twice with sterile, warm (37 °C), calcium-containing (at a concentration of 1,0-1,8 mM), phenol red-free, HBSS, and the fluorescein leakage should be immediately measured.
kategorija 2(A)
Concurrent negative (NC) and positive controls (PC) should be used in each run to demonstrate that monolayer integrity (NC) and sensitivity of the cells (PC) are within a defined historical acceptance range.
nejedka/lažje dražilna snov
The suggested PC chemical is Brij 35 (CAS No. 9002-92-0) at 100 mg/ml.
nejedka/lažje dražilna snov
This concentration should give approximately 30 % fluorescein leakage (acceptable range 20-40 % fluorescein leakage, i.e. damage to cell layer).
kategorija 2(B)
The suggested NC chemical is calcium-containing (at a concentration of 1,0-1,8 mM), phenol red-free, HBSS (untreated, blank control).
Na podlagi rezultatov očesne študije in vivo na kuncih (OECD TG 405, PM B.5) in v skladu z GHS ZN in CLP EU.
A maximum leakage control should also be included in each run to allow for the calculation of FL20 values.
Na podlagi rezultatov, pridobljenih s FL (protokol INVITTOX št. 71(6)).
Maximum leakage is determined using a control insert without cells.
B.62 Kometni preskus in vivo v alkalnih pogojih pri sesalcih
Determination of fluorescein permeability
Ta preskusna metoda (PM) je enakovredna Smernici za preskušanje (TG) OECD 489 (2016).