natgillin/translations-raw huggingface dataset:
Samtidige negative (NC) og positive kontroller (PC) bruges til at påvise, at monolagets integritet (NC) og cellernes følsomhed (PC) ligger inden for et forudfastsat acceptinterval.
25 - 250
Det foreslåede PC-kemikalie er Brij 35 (CAS-nr.
Methyldibromo glutaronitrile
9002-92-0) ved 100 mg/ml.
Sensitiser (strong)
Denne koncentration giver en fluoresceinudsivning på omkring 30 % (acceptabelt interval 20-40 % fluoresceinudsivning, dvs. skader på cellelaget).
20 - 100
Det foreslåede NC-kemikalie er calciumholdig (med en koncentration på 1,0-1,8 mM), phenolrødt-fri HBSS (ubehandlet, blindprøve).
ethylaminophenol sulfate
Der medtages også en maksimal udsivning i hver kørsel for at gøre det muligt at beregne FL20-værdier.
20 - 200
Den maksimale udsivning bestemmes ved hjælp af en kontrolindsats uden celler.
2,4-Dinitro-chlorobenzene
Bestemmelse af fluoresceinpermeabilitet
Sensitiser (extreme)
Målingerne foretages i et spektrofluorometer ved excitations- og emissionsbølgelængder på henholdsvis 485 nm og 530 nm.
5 - 20
Spektrofluorometerets følsomhed indstilles, så man opnår den højeste numeriske forskel mellem det maksimale FL (indsats uden celler) og det minimale FL (indsats med sammenflydende monolag behandlet med NC).
The in vivo hazard (and potency) predictions are based on LLNA data (13).
På grund af forskelle mellem de anvendte spektrofluorometre anbefales det at anvende en følsomhed, som giver en fluorescensintensitet > 4000 ved kontrol af den maksimale fluoresceinudsivning.
The in vivo potency is derived using the criteria proposed by ECETOC (24).
Den maksimale FL-værdi må ikke være højere end 9999.
A KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the provisions of paragraphs 9 and 11 of this test method.
Den maksimale intensitet af fluorescensudsivningen bør ligge inden for det anvendte spektrofluorometers lineære rækkevidde.
Based on the historical observed values (12).
Fortolkning af resultater og forudsigelsesmodel
QUALITY CONTROL OF LUMINESCENCE MEASUREMENTS
Mængden af FL er proportional med de kemikalieinducerede skader på tight junctions.
Basic experiment for ensuring optimal luminescence measurements in the KeratinoSens™ assay
FL-procenten for hver af de testede koncentrationer af kemikaliet beregnes ud fra de opnåede FL-værdier for testkemikaliet med henvisning til FL-værdier fra NC (aflæsning for sammenflydende monolag af celler behandlet med NC) og kontrollen for den maksimale udsivning (aflæsning for FL-mængden gennem en indsats uden celler).
The following three parameters are critical to ensure obtaining reliable results with the luminometer:
Den gennemsnitlige maksimale fluorescensintensitet = x
having a sufficient sensitivity giving a stable background in control wells;
Den gennemsnitlige maksimale procentvise fluorescensintensitet (NC) = y
having no gradient over the plate due to long reading times; and
Den gennemsnitlige 100 %-udsivning opnås ved at trække den gennemsnitlige 0 %-udsivning fra den gennemsnitlige maksimale udsivning,
having no light contamination in adjacent wells from strongly active wells.
dvs. x – y = z
Prior to testing it is recommended to ensure having appropriate luminescence measurements, by testing a control plate set-up as described below (triplicate analysis).
Den procentvise udsivning for hver af de fastsatte doser opnås ved at trække 0 %-udsivningen fra den gennemsnitlige fluorescensintensitet for de tre replikataflæsninger (m) og dividere denne værdi med 100 %-udsivningen, dvs. % FL = [ (m-y) / z] × 100 %, hvor:
Plate setup of first training experiment
m middelværdien for fluorescensintensiteten for de tre replikatmålinger for den pågældende koncentration
Blank
% FL procentdelen af fluorescein, der siver gennem cellelaget
EGDMA Ethylene glycol dimethacrylate (CAS No.: 97-90-5) a strongly inducing chemical
Følgende ligning til beregning af den kemiske koncentration, der forårsager 20 % FL, anvendes:
CA Cinnamic aldehyde, positive reference (CAS No.: 104-55-2)
FLD = [ (A-B) / (C-B)] × (MC – MB) + MB
The quality control analysis should demonstrate:
hvor
a clear dose-response in row D, with the Imax > 20 fold above background (in most cases Imax values between 100 and 300 are reached);
D % hæmning
no dose-response in row C and E (no induction value above 1,5 (ideally not above 1,3) due to possible light contamination especially next to strongly active wells in the EGDMA row;
A % skader (20 % fluoresceinudsivning)
no statistically significant difference between the rows A, B, C, E, F and G. (i.e. no gradient over plate); and
B % fluoresceinudsivning < A
variability in any of the rows A, B, C, E, F and G and in the DMSO wells in row H should be below 20 % (i.e. stable background).
C % fluoresceinudsivning > A
B.61 Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants
koncentration (mg/ml) af C
This test method (TM) is equivalent to OECD test guideline (TG) 460 (2012).
koncentration (mg/ml) af B
The Fluorescein Leakage (FL) test method is an in vitro test method that can be used under certain circumstances and with specific limitations to classify chemicals (substances and mixtures) as ocular corrosives and severe irritants, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Category 1), Regulation (EC) No1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) [19] (Category 1), and the U.S. Environmental Protection Agency (EPA) (Category I) (1)(2).
Tærskelværdien for FL20 for forudsigelse af, hvorvidt kemikalier er øjenætsende/stærkt øjenirriterende er anført nedenfor:
For the purpose of this test method, severe ocular irritants are defined as chemicals that cause tissue damage in the eye following test chemical administration that is not reversible within 21 days or causes serious physical decay of vision, while ocular corrosives are chemicals that cause irreversible tissue damage to the eye.
C&L: klassificering og mærkning
These chemicals are classified as UN GHS Category 1, EU CLP Category 1, or U.S. EPA Category I.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 use as part of a tiered testing strategy for regulatory classification and labelling.
Kategori I
Thus, the FL is recommended as an initial step within a Top-Down approach to identify ocular corrosives/severe irritants, specifically for limited types of chemicals (i.e. water soluble substances and mixtures) (3)(4).
FL-forsøgsmetoden anbefales kun til identifikation af vandopløselige øjenætsende stoffer og stærkt øjenirriterende stoffer (UN GHS-kategori 1, EU CLP-kategori 1, U.S. EPA-kategori I) (se punkt 1 og 10).
It is currently generally accepted that, in the foreseeable future, no single in vitro eye irritation test will be able to replace the in vivo eye test (TM B.5 (5)) to predict across the full range of irritation for different chemical classes.
Med henblik på at identificere vandopløselige kemikalier (stoffer og blandinger) (3) (6) (7), som “medfører alvorlig øjenskade” (UN GHS/EU CLP-kategori 1), eller som er “øjenætsende eller stærkt øjenirriterende” (U.S. EPA-kategori I), skal testkemikaliet inducere en FL20-værdi på ≤ 100 mg/ml.
However, strategic combinations of several alternative test methods within a (tiered) testing strategy may be able to replace the in vivo eye test (4).
Accept af resultater
The Top-Down approach (4) is designed to be used when, based on existing information, a chemical is expected to have high irritancy potential.
Den gennemsnitlige maksimale værdi for fluoresceinudsivning (x) skal være højere end 4000 (se punkt 31), den gennemsnitlige 0 %-udsivning (y) skal være mindre end eller lig med 300, og 100 %-udsivningen (z) skal ligge mellem 3700 og 6000.
Based on the prediction model detailed in paragraph 35, the FL test method can identify chemicals within a limited applicability domain as ocular corrosives/severe irritants (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I) without any further testing.
En test anses for acceptabel, hvis den positive kontrol medførte 20 % til 40 % skade på cellelaget (målt som % fluoresceinudsivning).
The same is assumed for mixtures although mixtures were not used in the validation.
For hver analyseserie bør data fra de enkelte replikatbrønde (f.eks. værdier for fluorescensaktivitet og beregnede procentvise FL-data for hvert testkemikalie samt dets klassificering) fremlægges i tabelform.
Therefore, the FL test method may be used to determine the eye irritancy/corrosivity of chemicals, following the sequential testing strategy of TM B.5 (5).
Desuden bør gennemsnit og standardafvigelse for individuelle gentagne målinger i hver kørsel rapporteres.
However, a chemical that is not predicted as ocular corrosive or severe 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 false negative ocular corrosives/severe 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).
kemikaliets CAS-nummer, hvis dette er kendt
The purpose of this test method is to describe the procedures used to evaluate the potential ocular corrosivity or severe irritancy of a test chemical as measured by its ability to induce damage to an impermeable confluent epithelial monolayer.
renhed og sammensætning af stoffet eller blandingen (i vægtprocent) i det omfang, disse oplysninger er tilgængelige
The integrity of trans-epithelial permeability is a major function of an epithelium such as that found in the conjunctiva and the cornea.
fysisk-kemiske egenskaber, som er relevante for undersøgelsen (f.eks. fysisk tilstand, flygtighed, pH, stabilitet, opløselighed i vand, kemikaliegruppe)
Trans-epithelial permeability is controlled by various tight junctions.
eventuel behandling af test-/kontrolkemikalier inden testen (f.eks. opvarmning, formaling)
Increasing the permeability of the corneal epithelium in vivo has been shown to correlate with the level of inflammation and surface damage observed as eye irritation develops.
opbevaringsforhold.
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.
Begrundelse for den anvendte forsøgsmetode og protokol
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.
bør indeholde overvejelser vedrørende anvendelsesområde og forsøgsmetodens begrænsninger.
Appendix 1 provides a diagram of MDCK cells grown on an insert membrane for the FL test method.
beskrivelse af det anvendte cellesystem, herunder ægthedscertifikatet og mycoplasmastatus for cellelinjen
Definitions are provided in Appendix 2.
detaljer om den anvendte testprocedure
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).
de(n) anvendte koncentration(er) af testkemikaliet
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.
varighed af eksponering for testkemikaliet
2A/2B, no category; EU CLP Cat.
varighed af inkuberingen med fluorescein
2, no category; US EPA Cat.
beskrivelse af de anvendte vurderingskriterier
II/III/IV), as demonstrated by the validation study (3) (7).
reference til historiske data for modellen (f.eks. negative og positive kontroller, referencekemikalier, hvis dette er relevant)
The test method is only applicable to water soluble chemicals (substances and mixtures).
oplysninger om den tekniske kompetence, som laboratoriet har dokumenteret.
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).
Opstilling i tabelform af data for hvert enkelt testkemikalie, for kontrollerne i hver kørsel og fra hver flergangsbestemmelse (inklusive individuelle resultater, gennemsnit og standardafvigelser)
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).
de(n) afledte klassificering(er) med henvisning til den anvendte forudsigelsesmodel og/eller beslutningskriterier
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.
Bør omfatte overvejelser vedrørende et usikkert resultat (punkt 35: FL20 > 100 mg/ml) og yderligere undersøgelser
1 or EPA Cat.
Konklusioner
I.
Kapitel B.5 i dette bilag, Akut øjenirritation/-ætsning.
The identified limitations for this test method exclude strong acids and bases, cell fixatives and highly volatile chemicals from the applicability domain.
Tilgængelig på: [http://ecvam-dbalm.jrc.ec.europa.eu/]
These chemicals have mechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponification or specific reactive chemistries.
OECD (2005), Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No. 34, OECD, Paris.
Other identified limitations for this method are based upon the results for the predictive capacity for coloured and viscous test chemical (7).
DIAGRAM OVER MDCK-CELLER DYRKET PÅ EN INDSATSMEMBRAN TIL FL-FORSØGSMETODEN
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.
Et sammenflydende lag MDCK-celler dyrkes på en semipermeabel membran fra en indsats.
Solid chemicals suspended in liquid have the propensity to precipitate out and the final concentration to cells can be difficult to determine.
Indsatserne placeres i plader med 24 brønde.
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).
Figur hentet fra: Wilkinson, P.J.
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).
EPA-kategori I
Other identified limitations of the FL test method are based on false negative and false positive rates.
Kemikalier, der forårsager ætsende virkning (irreversibel ødelæggelse af øjenvæv) eller cornea-beskadigelse eller -irritation, som persisterer i mere end 21 dage (2).
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.
EU CLP (forordning (EF) nr. 1272/2008 om klassificering, mærkning og emballering af stoffer og blandinger)
Chemical groups showing false positive and/or false negative results in the FL test method are not defined here.
Gennemfører FN's globalt harmoniserede system for klassificering af kemikalier (stoffer og blandinger) (GHS) i Den Europæiske Union (EU).
Certain technical limitations are specific to the MDCK cell culture.
Kan vurderes ved bestemmelse af den koncentration, hvor testkemikaliet forårsager 20 % af fluoresceinudsivningen gennem cellelaget.
The tight junctions that block the passage of the sodium-fluorescein dye through the monolayer are increasingly compromised with increasing cell passage number.
Fluoresceinudsivning
Incomplete formation of the tight junctions results in increased FL in the non-treated control.
Mængden af fluorescein, der passerer gennem cellelaget, målt med spektrofluorometri.
Therefore, a defined permissible maximal leakage in the non-treated controls is important (see paragraph 38: 0 % leakage).
De Forenede Nationers (FN) globalt harmoniserede system for klassificering og mærkning af kemikalier (GHS)
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.
System til klassificering af kemikalier (stoffer og blandinger) efter standardiserede typer og niveauer af fysisk fare, sundhedsfare og miljøfare med tilhørende kommunikationselementer såsom piktogrammer, signalord, risikosætninger, sikkerhedssætninger og sikkerhedsdatablade, således at oplysninger om kemikaliernes skadelige virkninger kan videreformidles med henblik på at beskytte mennesker (f.eks. arbejdsgivere, arbejdstagere, transportører, forbrugere og redningspersonel) og miljø.
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).
GHS-kategori 1
This test method will be updated accordingly as new information and data are considered.
Brugt i forbindelse med UN GHS som en blanding eller opløsning, der er sammensat af to eller flere stoffer, hvori de ikke reagerer.
For any laboratory initially establishing this assay, the proficiency chemicals provided in Appendix 3 should be used.
Ikke klassificeret
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.
kemikalie, der forårsager irreversibel vævsbeskadigelse i øjet; b) kemikalier, der er klassificeret som øjenirriterende stoffer i UN GHS-kategori 1, EU CLP-kategori 1 eller U.S.
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.
Stærkt øjenirriterende stof
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.
kemikalie, som forårsager vævsbeskadigelse i øjet efter påføring på øjets anteriore overflade, som ikke er reversibel inden for 21 dage efter påføring, eller som forårsager alvorlig fysisk synsnedsættelse; b) kemikalier, der er klassificeret som øjenirriterende stoffer i UN GHS-kategori 1, EPA-kategori I, EU CLP-kategori 1 eller U.S.
Tight and desmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal epithelium.
EPA-kategori I.
Loss of trans-epithelial impermeability, due to damaged tight junctions and desmosomal junctions, is one of the early events in chemical-induced ocular irritation.
For at sikre, at det er muligt at vurdere variabiliteten i den positive kontrolrespons over tid, bør den positive respons ikke være overdrevent stor.
The test chemical is applied to the confluent layer of cells grown on the apical side of the insert.
Kompetencekemikalier
A short 1 min exposure is routinely used to reflect the normal clearance rate in human exposures.
Undergruppe af listen over referencekemikalier, der kan anvendes af et uerfarent laboratorium til at dokumentere fortrolighed med den validerede forsøgsmetode.
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.
Forsøgsmetodens nøjagtighed (konkordans) indgår i vurderingen af dens relevans (8).
This allows more direct comparisons of the results with the chemical effects in humans.
Erstatningstest
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.
Test, der erstatter en test, som bruges rutinemæssigt og er godkendt til fareidentifikation og/eller risikovurdering, og som vurderes at sikre en tilsvarende eller bedre beskyttelse af menneskers og dyrs sundhed eller miljøet alt afhængigt af situationen i forhold til den godkendte test, i alle testsituationer og for alle kemikalier.
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.
Andel af alle positive/aktive kemikalier, der klassificeres korrekt ved testen.
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.
Det er et mål for nøjagtigheden af en forsøgsmetode, som giver kategoriske resultater, og er et vigtigt kriterium ved vurderingen af en forsøgsmetodes relevans (8).
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.
Frembringelse af vævsbeskadigelse i øjet eller alvorlig fysisk synsnedsættelse, som opstår efter påføring af et testkemikalie på øjets anteriore overflade, og som ikke er fuldt reversibel inden for 21 dage efter påføring.
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.
Ubehandlet prøve, som indeholder alle komponenterne fra et testsystem, herunder opløsningsmidlet eller bærestoffet, og som behandles sammen med de testkemikaliebehandlede prøver og andre kontrolprøver for at fastlægge basisresponsen for de prøver, som er behandlet med testkemikaliet opløst i det samme opløsningsmiddel eller bærestof.
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.
Andel af alle negative/inaktive kemikalier, der klassificeres korrekt ved testen.
The FL20 (mg/ml) value is used in the prediction model for identification of ocular corrosives and severe irritants (see paragraph 35).
Det er vigtigt at være opmærksom på, at en valideret forsøgsmetode måske ikke har tilstrækkelig ydeevne med hensyn til nøjagtighed og pålidelighed til at være acceptabel til det planlagte formål (8).
Preparation of the cellular monolayer
KOMPETENCEKEMIKALIER TIL FL-FORSØGSMETODEN
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).