natgillin/translations-raw huggingface dataset:
Cellernes levedygtighed er større end (>) 70 % ved den laveste koncentration med induktion af luciferaseaktivitet over 1,5 fold (dvs. ved den EC1,5-fastsættende koncentration).
However, further work preferably based on reliable human data is required to determine how KeratinoSensTM assay results can contribute to potency assessment (24) and to sub-categorisation of sensitisers according to UN GHS/CLP.
,5-værdien er mindre end (<) 1000 μM (eller < 200 μg/ml for testkemikalier uden defineret molekylvægt).
The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid.
Der findes en tilsyneladende generel dosisrespons for luciferaseinduktion (eller en bifasisk respons som nævnt i punkt 33).
The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers (25) (26).
Hvis alle de tre første betingelser er opfyldt i en given gentagelse, men der ikke kan observeres en klar dosisrespons for luciferaseinduktionen, bør resultatet af den pågældende gentagelse betragtes som usikkert, og der kan være behov for yderligere undersøgelser (figur 1).
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated (27).
Desuden bør et negativt resultat opnået med koncentrationer < 1000 μM (eller < 200 μg/ml for testkemikalier uden defineret molekylvægt) også betragtes som usikkert (jf. punkt 11).
This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.
Figur 1
Test chemicals are considered positive in the KeratinoSens™ assay if they induce a statistically significant induction of the luciferase activity above a given threshold (i.e. > 1,5 fold or 50 % increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 μM and at a concentration at which the cellular viability is above 70 % (9) (12)).
Forudsigelsesmodel, der anvendes i KeratinoSensTM-assayet.
For this purpose, the maximal fold induction of the luciferase activity over solvent (negative) control (Imax) is determined.
En KeratinoSensTM-forudsigelse bør overvejes inden for rammerne af en IATA og i overensstemmelse med bestemmelserne i punkt 9 og 11.
Furthermore, since cells are exposed to series of concentrations of the test chemicals, the concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1,5 value) should be interpolated from the dose-response curve (see paragraph 32 for calculations).
I sjældne tilfælde kan testkemikalier, der inducerer luciferaseaktivitet meget tæt på de cytotoksiske niveauer, være positive i nogle gentagelser ved ikke-cytotoksiske niveauer (dvs. ved EC1,5-bestemmende koncentrationer under (<) IC30), og i andre gentagelser kun ved cytotoksiske niveauer (dvs. ved EC1,5-bestemmende koncentrationer over (>) IC30).
Finally, parallel cytotoxicity measurements should be conducted to assess whether luciferase activity induction levels occur at sub-cytotoxic concentrations.
Sådanne testkemikalier testes på ny med en smallere dosisresponsanalyse under anvendelse af en lavere fortyndingsfaktor (f.eks. en 1,33 eller √2 (= 1,41) fold fortynding mellem brøndene) for at fastslå, om induktionen har fundet sted ved cytotoksiske niveauer eller ej (9).
Prior to routine use of the ARE-Nrf2 luciferase assay that adheres to this test method, laboratories should demonstrate technical proficiency, using the ten Proficiency Substances listed in Appendix 2.
fysisk udseende, vandopløselighed, DMSO-opløselighed, molekylvægt og yderligere relevante fysisk-kemiske egenskaber i den udstrækning, det er muligt
Performance standards (PS) (28) are available to facilitate the validation of new or modified in vitro ARE-Nrf2 luciferase test methods similar to the KeratinoSens™ assay and allow for timely amendment of this test method for their inclusion.
fysisk udseende, vandopløselighed, DMSO-opløselighed og yderligere relevante fysisk-kemiske egenskaber i den udstrækning, det er muligt
Mutual Acceptance of Data (MAD) according to the OECD agreement will only be guaranteed for test methods validated according to the PS, if these test methods have been reviewed and included in the corresponding test guideline by OECD.
fysisk udseende, vandopløselighed, DMSO-opløselighed, molekylvægt og yderligere relevante fysisk-kemiske egenskaber i den udstrækning, det er muligt og relevant
Currently, the only method covered by this test method is the scientifically valid KeratinoSensTM assay (9) (12) (13) (14).
Negativ kontrol (bærestofkontrol)
The Standard Operating Procedures (SOP) for the KeratinoSensTM assay is available and should be employed when implementing and using the test method in the laboratory (15).
kemisk identifikation, f.eks. IUPAC- eller CAS-navn(e), CAS-nummer/numre og/eller andre identifikatorer
Laboratories willing to implement the test method can obtain the recombinant cell line used in the KeratinoSensTM assay by establishing a licence agreement with the test method developer.
fysisk udseende, molekylvægt og yderligere relevante fysisk-kemiske egenskaber ved brug af andre negative kontroller/bærestoffer end dem, der er nævnt i forsøgsmetoden, i det omfang det er muligt
The following paragraphs provide with a description of the main components and procedures of the ARE-Nrf2 luciferase test method.
begrundelse for valg af opløsningsmiddel for de enkelte testkemikalier
Preparation of the keratinocyte cultures
betingelser for forsøgsmetoden
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element should be used (e.g. the KeratinoSens™ cell line).
beskrivelse af forsøgsmetoden
Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
anvendt cellelinje, opbevaringsforhold og kilde (f.eks. det anlæg, som den er hentet fra)
Cells from this original stock can be propagated up to a maximum passage number (i.e. 25 in the case of KeratinoSensTM) and are employed for routine testing using the appropriate maintenance medium (in the case of KeratinoSensTM this represents DMEM containing serum and Geneticin).
antal generationer og graden af konfluens for de celler, der anvendes til test
For testing, cells should be 80-90 % confluent, and care should be taken to ensure that cells are never grown to full confluence.
anvendt celletællingsmetode til udsåning inden testen, og de foranstaltninger, der er truffet for at sikre en ensartet fordeling af celleantal (jf. punkt 20)
One day prior to testing cells are harvested, and distributed into 96-well plates (10000 cells/well in the case of KeratinoSensTM).
anvendt luminometer (f.eks. model), herunder instrumentindstillinger, det anvendte luciferasesubstrat og dokumentation for hensigtsmæssige luminescensmålinger baseret på kontrolprøven, der er beskrevet i tillæg 3
Attention should be paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells.
den procedure, der anvendes til at påvise laboratoriets kompetence til at anvende forsøgsmetoden (f.eks. ved test af kompetencestoffer) eller til at påvise reproducerbare resultater af forsøgsmetoden over tid.
If this is not the case, this step may give raise to high well-to-well variability.
Testprocedure
For each repetition, three replicates are used for the luciferase activity measurements, and one parallel replicate used for the cell viability assay.
antal anvendte gentagelser og replikater
Preparation of the test chemical and control substances
koncentrationer af testkemikaliet, tilsætningsprocedure og eksponeringstid (hvis anderledes end anbefalet)
The test chemical and control substances are prepared on the day of testing.
opstilling i tabelform af Imax, EC1,5 og levedygtighedsværdier (dvs. IC50, IC30), som er opnået for testkemikaliet og for den positive kontrol for hver gentagelse samt middelværdierne (Imax: gennemsnit, EC1,5 og levedygtighedsværdier: geometrisk middelværdi) og standardafvigelse beregnet ved hjælp af data fra de enkelte gentagelser samt en angivelse af vurderingen af testkemikaliet i henhold til forudsigelsesmodellen
For the KeratinoSensTM assay, test chemicals are dissolved in dimethyl sulfoxide (DMSO) to the final desired concentration (e.g. 200 mM).
opnået variationskoefficient ved luminescensaflæsningerne for den negative kontrol for hvert forsøg
The DMSO solutions can be considered self-sterilising, so that no sterile filtration is needed.
graf, der viser dosis/respons-kurver for induktion af luciferaseaktivitet og levedygtighed
Test chemical not soluble in DMSO is dissolved in sterile water or culture medium, and the solutions sterilised by e.g. filtration.
beskrivelse af eventuelle andre relevante observationer, hvis det er relevant.
For a test chemical which has no defined molecular weight (MW), a stock solution is prepared to a default concentration (40 mg/mL or 4 % (w/v)) in the KeratinoSensTM assay.
diskussion af de resultater, der er opnået med KeratinoSensTM-assayet
In case solvents other than DMSO, water or the culture medium are used, sufficient scientific rationale should be provided.
Kapitel B.50 i dette bilag: Hudsensibilisering: Assay på lokale lymfeknuder: DA.
Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
Recommendation on the KeratinoSensTM assay for skin sensitisation testing, 42 pp.
It undergoes the same dilution as described for the master concentrations in paragraph 22, so that the final negative (solvent) control concentration is 1 %, known not to affect cell viability and corresponding to the same concentration of DMSO found in the tested chemical and in the positive control.
DB-ALM (INVITTOX) (2013) Protocol 155: KeratinoSensTM., 17 pp.
The positive control used in the case of the KeratinoSensTM assay is cinnamic aldehyde (CAS No. 14371-10-9, ≥ 98 % purity), for which a series of 5 master concentrations ranging from 0,4 to 6,4 mM are prepared in DMSO (from a 6,4 mM stock solution) and diluted as described for the master concentrations in paragraph 22, so that the final concentration of the positive control range from 4 to 64 μM.
Den er et mål for forsøgsmetodens ydeevne og et af aspekterne af »relevans«.
Application of the test chemical and control substances
Udtrykket benyttes ofte som synonym for »konkordans«, forstået som den andel af korrekte udfald, forsøgsmetoden fører til (29).
For each test chemical and positive control substance, one experiment is needed to derive a prediction (positive or negative), consisting of at least two independent repetitions containing each three replicates (i.e. n = 6).
Antioxidantresponselement (også kaldet EpRE, electrofilresponselement) — et responselement, der findes i upstream-promotorområdet for mange cytobeskyttende gener og fase II-gener.
In case of discordant results between the two independent repetitions, a third repetition containing three replicates should be performed (i.e. n = 9).
Når det aktiveres af Nfr2, igangsætter det transskriptionsinduktionen for disse gener.
Each independent repetition is performed on a different day with fresh stock solution of test chemicals and independently harvested cells.
Interpoleret koncentration for en 1,5 fold luciferaseinduktion.
Cells may come from the same passage however.
Koncentration, der medfører nedsættelse af cellelevedygtigheden med 30 %.
After seeding as described in paragraph 20, cells are grown for 24 hours in the 96-wells microtiter plates.
Koncentration, der medfører nedsættelse af cellelevedygtigheden med 50 %.
The medium is then removed and replaced with fresh culture medium (150 μl culture medium containing serum but without Geneticin in the case of KeratinoSensTM) to which 50 μl of the 25 fold diluted test chemical and control substances are added.
Maksimal induktionsfaktor for luciferaseaktivitet sammenlignet med opløsningsmiddelkontrollen (den negative kontrol) målt for en hvilken som helst koncentration af testkemikaliet.
At least one well per plate should be left empty (no cells and no treatment) to assess background values.
Kelch-lignende ECH-associeret protein 1 — et sensorprotein, der kan regulere Nrf2-aktiviteten.
The treated plates are then incubated for about 48 hours at 37 ± 1 °C in the presence of 5 % CO2 in the KeratinoSensTM assay.
Under ikke-inducerede betingelser er Keap1-sensorproteinet rettet mod Nrf2-transskriptionsfaktoren ubitquitinylering og proteolytisk nedbrydning i proteasomet.
Care should be taken to avoid evaporation of volatile test chemicals and cross-contamination between wells by test chemicals by e.g. covering the plates with a foil prior to the incubation with the test chemicals.
Den bedømmes ved beregning af reproducerbarhed inden for og mellem laboratorier og repeterbarhed inden for laboratorier (29).
the use of a luciferase substrate with sufficient light output to ensure sufficient sensitivity and low variability.
Andel af alle positive/aktive kemikalier, der klassificeres korrekt ved forsøgsmetoden.
After the 48 hour exposure time with the test chemical and control substances in the KeratinoSensTM assay, cells are washed with a phosphate buffered saline, and the relevant lysis buffer for luminescence readings added to each well for 20 min at room temperature.
Replikat, der indeholder alle bestanddele af et testsystem, undtagen testkemikaliet, men inklusive det anvendte opløsningsmiddel.
In case alternative settings are used, e.g. depending on the model of luminometer used, these should be justified.
Det anvendes til at bestemme basisresponsen for prøver behandlet med testkemikaliet, der er opløst i det samme opløsningsmiddel.
Furthermore, a glow substrate may also be used provided that the quality control experiment of Appendix 3 is successfully fulfilled.
Andel af alle negative/inaktive kemikalier, der klassificeres korrekt ved forsøgsmetoden.
Cytotoxicity Assessment
Det er et mål for nøjagtigheden af en forsøgsmetode, som giver kategoriske resultater, og er et vigtigt kriterium ved vurderingen af en forsøgsmetodes relevans (29).
For the KeratinoSensTM cell viability assay, medium is replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells incubated for 4 hours at 37 °C in the presence of 5 % CO2.
En forsøgsmetode har aldrig absolut gyldighed, men kun i forbindelse med et bestemt formål (29).
The MTT medium is then removed and cells are lysed (e.g. by adding 10 % SDS solution to each well) overnight.
In vitro-hudsensibilisering: ARE-Nrf2-luciferase-forsøgsmetoden
After shaking, the absorption is measured at i.e. 600 nm with a photometer.
Før rutinemæssig brug af denne forsøgsmetode bør laboratorierne godtgøre deres tekniske kompetence ved at opnå den forventede KeratinoSensTM-forudsigelse på korrekt vis for de 10 kompetencestoffer, der anbefales i tabel 1, og ved at opnå de EC1,5- og IC50-værdier, der falder ind under de respektive referenceområder for 1,5 ud af de 10 kompetencestoffer.
The following parameters are calculated in the KeratinoSensTM assay:
Disse kompetencestoffer er udvalgt således, at de repræsenterer responsområdet for hudsensibiliseringsfare.
the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
Andre udvælgelseskriterier er kommerciel tilgængelighed, tilgængelighed af in vivo-referencer af høj kvalitet og tilgængelighed af in vitro-data af høj kvalitet for KeratinoSensTM-assayet.
the EC1,5 value representing the concentration for which induction of luciferase activity is above the 1,5 fold threshold (i.e. 50 % enhanced luciferase activity) was obtained; and
Anbefalede stoffer til godtgørelse af teknisk kompetence i anvendelse af KeratinoSens™-assayet
the IC50 and IC30 concentration values for 50 % and 30 % reduction of cellular viability.
Kompetencestoffer
Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
In vivo-forudsigelse [1]
Equation 1:
KeratinoSens™ Forudsigelse [2]
Fold induction
,5 (μM)-referenceinterval [3]
Lsample
(μM)-referenceinterval [3]
Lblank
Ikke-sensibiliserende
Lsolvent
Salicylsyre
where
Mælkesyre
L sample
Kanelalkohol
is the luminescence reading in the test chemical well
Sensibiliserende (svagt)
L blank
25-175
is the luminescence reading in the blank well containing no cells and no treatment
Ethylenglycoldimethacrylat
L solvent
5-125
is the average luminescence reading in the wells containing cells and solvent (negative) control
ercaptobenzothiazol
,5 is calculated by linear interpolation according to Equation 2, and the overall EC1,5 is calculated as the geometric mean of the individual repetitions.
Sensibiliserende (moderat)
Equation 2:
25-250
is the lowest concentration in μM with > 1,5 fold induction
Methyldibromglutaronitril
is the highest concentration in μM with < 1,5 fold induction
Sensibiliserende (stærkt)
is the fold induction measured at the lowest concentration with > 1,5 fold induction (mean of three replicate wells)
20-100
is the fold induction at the highest concentration with < 1,5 fold induction (mean of three replicate wells)
ethylaminophenolsulfat
Viability is calculated by Equation 3:
20-200
Equation 3:
2,4-dinitro-chlorbenzen
Viability
Sensibiliserende (ekstremt)
Vsample
5-20
Vblank
Prognoser for in vivo-fare og (styrke) er baseret på LLNA-data (13).
Vsolvent
In vivo-styrken er beregnet ud fra de kriterier, der foreslås af ECETOC (24).
V sample
Man bør overveje en KeratnoSens™-forudsigelse inden for rammerne af en IATA og i overensstemmelse med bestemmelserne i punkt 9 og 11 i denne forsøgsmetode.
is the MTT-absorbance reading in the test chemical well
På baggrund af de historiske observerede værdier (12).
V blank
KVALITETSKONTROL AF LUMINESCENSMÅLINGER
is the MTT-absorbance reading in the blank well containing no cells and no treatment
Grundlæggende forsøg til sikring af optimale luminescensmålinger i KeratinoSens™-assayet
V solvent
Følgende tre parametre er afgørende for at sikre opnåelsen af pålidelige resultater med luminometeret:
is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
tilstrækkelig følsomhed, der giver en stabil baggrund i kontrolbrøndene
and IC30 are calculated by linear interpolation according to Equation 4, and the overall IC50 and IC30 are calculated as the geometric mean of the individual repetitions.
ingen gradient over pladen som følge af lange aflæsningstider
Equation 4:
ingen lysforurening af hosliggende brønde fra stærkt aktive brønde.
X is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Inden testen anbefales det at sikre passende luminescensmålinger ved at teste en kontrolpladeopstilling som beskrevet nedenfor (tredobbelt analyse).
is the lowest concentration in μM with > x % reduction in viability
Pladeopstilling for første kontroltest
is the highest concentration in μM with < x % reduction in viability
Blindprøve
is the % viability at the lowest concentration with > x % reduction in viability
EGDMA Ethylenglycoldimethacrylat (CAS-nr.: 97-90-5, et kraftigt inducerende kemikalie
is the % viability at the highest concentration with < x % reduction in viability
CA kanelaldehyd, positivreference (CAS-nr.: 104-55-2)
For each concentration showing > 1,5 fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p < 0,05).
Analysen af kvalitetskontrollen bør godtgøre:
The lowest concentration with > 1,5 fold luciferase activity induction is the value determining the EC1,5 value.
en klar dosisrespons i række D, med Imax > 20 fold over baggrundsværdien (i de fleste tilfælde opnås Imax-værdier mellem 100 og 300)
It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30 % reduction in cellular viability at the EC1,5 determining concentration.
ingen dosisrespons i række C og E (ingen induktionsværdi over 1,5 (helst ikke over 1,3) som følge af eventuel let baggrundsforurening, navnlig ved siden af meget aktive brønde i EGDMA-rækken
It is recommended that data are visually checked with the help of graphs.
ingen statistisk signifikant forskel mellem række A, B, C, E, F og G (dvs. ingen gradient over pladen)
If no clear dose-response curve is observed, or if the dose-response curve obtained is biphasic (i.e. crossing the threshold of 1,5 twice), the experiment should be repeated to verify whether this is specific to the test chemical or due to an experimental artefact.
variationen bør ikke være under 20 % i nogen af rækkerne A, B, C, E, F og G og DMSO-brøndene i række H (dvs. stabil baggrund).
In case the biphasic response is reproducible in an independent experiment, the lower EC1,5 value (the concentration when the threshold of 1,5 is crossed the first time) should be reported.
B.61 Fluoresceinudsivnings-forsøgsmetoden til identifikation af øjenætsende stoffer og stærkt øjenirriterende stoffer
In the rare cases where a statistically non-significant induction above 1,5 fold is observed followed by a higher concentration with a statistically significant induction, results from this repetition are only considered as valid and positive if the statistically significant induction above the threshold of 1,5 was obtained for a non-cytotoxic concentration.
Denne forsøgsmetode svarer til OECD's testvejledning (Test Guideline (TG)) 460 (2012).
Finally, for test chemicals generating a 1,5 fold or higher induction already at the lowest test concentration of 0,98 μM, the EC1,5 value of < 0,98 is set based on visual inspection of the dose-response curve.
Ved denne forsøgsmetode defineres stærkt øjenirriterende stoffer som kemikalier, der forårsager vævsødelæggelse i øjet efter indgivelse af testkemikaliet, som ikke er reversible inden for 21 dage, eller som forårsager alvorlig fysisk synsnedsættelse, mens øjenætsende stoffer er kemikalier, som forårsager irreversibel vævsødelæggelse.
The following acceptance criteria should be met when using the KeratinoSensTM assay.
Det betyder, at FL anbefales som første trin i en top-down-tilgang til identifikation af øjenætsende stoffer/stærkt øjenirriterende stoffer specifikt for begrænsede typer af kemikalier (dvs. vandopløselige stoffer og blandinger) (3) (4).
First, the luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1,5 (e.g. using a T-test) in at least one of the tested concentrations (from 4 to 64 μM).
I dag er det generelt accepteret, at der i den nærmeste fremtid ikke vil være en enkelt in vitro-øjenirritationstest, som vil kunne erstatte in vivo-øjentesten (TM B.5 (5)) til forudsigelse af hele spektret af irritation for forskellige kemikaliegrupper.
Second, the EC1,5 value should be within two standard deviations of the historical mean of the testing facility (e.g. between 7 μM and 30 μM based on the validation dataset) which should be regularly updated.
Strategiske kombinationer af flere alternative forsøgsmetoder i en (trinvis) teststrategi kan dog muligvis erstatte in vivo-øjentesten (4).
In addition, the average induction in the three replicates for cinnamic aldehyde at 64 μM should be between 2 and 8.
Det samme forudsættes for blandinger, selv om blandinger ikke blev anvendt ved valideringen.
If the variability is higher, results should be discarded.