natgillin/translations-raw huggingface dataset:
However, the accuracy values given here for the KeratinoSensTM assay as a stand-alone test method are only indicative since the test method should be considered in combination with other sources of information in the context of an IATA and in accordance with the provisions of paragraph 9 above.
Soovitatav negatiivse kontrolli kemikaal on kaltsiumit (kontsentratsioonis 1,0–1,8 mM) sisaldav fenoolpunaseta HBSS (ilma uuritava kemikaalita, tühikatse kontroll).
Furthermore when evaluating non-animal methods for skin sensitisation, it should be kept in mind that the LLNA as well as other animal tests, may not fully reflect the situation in the species of interest i.e. humans.
Igasse katseseeriasse peab samuti olema lisatud maksimumlekke kontroll, et oleks võimalik arvutada FL20 väärtusi.
The term “test chemical” is used in this test method to refer to what is being tested and is not related to the applicability of the ARE-Nrf2 luciferase test method to the testing of substances and/or mixtures.
Maksimumleke määratakse, kasutades kontrollina rakkudeta vahemembraani.
On the basis of the current data available the KeratinoSensTM assay was shown to be applicable to test chemicals covering a variety of organic functional groups, reaction mechanisms, skin sensitisation potency (as determined with in vivo studies) and physico-chemical properties (9) (12) (13) (14).
Fluorestseiini suhtes läbilaskvuse määramine
Mainly mono-constituent substances were tested, although a limited amount of data also exist on the testing of mixtures (20).
Kohe pärast uuritava kemikaali ja kontrolli kemikaalide eemaldamist lisatakse vahemembraanidele (nt Millicell-HA) 400 μl naatriumfluorestseiini lahust kontsentratsioonis 0,1 mg/ml (0,01 %, mass/ruumala) kaltsiumit (kontsentratsioonis 1,0–1,8 mM) sisaldavas fenoolpunaseta HBSSis.
The test method is nevertheless technically applicable to the testing of multi-constituent substances and mixtures.
Rakukultuure hoitakse 30 minutit toatemperatuuril.
However, before use of this test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose.
Pärast fluorestseiiniga inkubeerimise lõppu eemaldatakse igast kannust ettevaatlikult vahemembraanid.
Moreover, when testing multi-constituent substances or mixtures, consideration should be given to possible interference of cytotoxic constituents with the observed responses.
Iga filtrit kontrollitakse visuaalselt ja registreeritakse iga kahjustus, mis võib olla tekkinud käsitsemise ajal.
The test method is applicable to test chemicals soluble or that form a stable dispersion (i.e. a colloid or suspension in which the test chemical does not settle or separate from the solvent into different phases) either in water or DMSO (including all of the test chemical components in the case of testing a multi-constituent substance or a mixture).
Läbi monokihi ja vahemembraani lekkinud fluorestseiini kogus mõõdetakse lahusest, mis jääb kannudesse pärast vahemembraani eemaldamist.
Test chemicals that do not fulfil these conditions at the highest final required concentration of 2000 μM (cf. paragraph 22) may still be tested at lower concentrations.
Mõõdetakse spektrofluoromeetriga ergastuse ja emissiooni lainepikkustel vastavalt 485 nm ja 530 nm.
In such a case, results fulfilling the criteria for positivity described in paragraph 39 could still be used to support the identification of the test chemical as a skin sensitiser, whereas a negative result obtained with concentrations < 1000 μM should be considered as inconclusive (see prediction model in paragraph 39).
Spektrofluoromeetri tundlikkus tuleb reguleerida nii, et tekiks suurim arvuline erinevus fluorestseiini maksimumlekke (rakkudeta membraan) ja miinimumlekke (negatiivse kontrolliga töödeldud rakkude konfluentse monokihiga membraan) vahel.
In general substances with a LogP of up to 5 have been successfully tested whereas extremely hydrophobic substances with a LogP above 7 are outside the known applicability of the test method (14).
Kuna kasutatavate spektrofluoromeetrite vahel on erinevusi, soovitatakse kasutada tundlikkust, mille korral fluorestseiini maksimumlekke kontrollis on tekkiva fluorestsentsi intensiivsus > 4000.
For substances having a LogP falling between 5 and 7, only limited information is available.
Fluorestseiini maksimumlekke väärtus ei tohi olla suurem kui 9999.
Negative results should be interpreted with caution as substances with an exclusive reactivity towards lysine-residues can be detected as negative by the test method.
Fluorestseiini maksimumlekke intensiivsus peab jääma kasutatava spektrofluoromeetri lineaarsusvahemikku.
Furthermore, because of the limited metabolic capability of the cell line used (21) and because of the experimental conditions, pro-haptens (i.e. chemicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens (i.e. chemicals activated by auto-oxidation) in particular with a slow oxidation rate may also provide negative results.
Tulemuste tõlgendamine ja prognoosimudel Fluorestseiini lekke kogus on proportsionaalne kemikaali põhjustatud tiheliiduste kahjustusega.
Test chemicals that do not act as a sensitiser but are nevertheless chemical stressors may lead on the other hand to false positive results (14).
Kemikaali iga uuritud kontsentratsiooni kohta arvutatakse fluorestseiini lekke protsent, lähtudes fluorestseiini lekke väärtustest, mis saadakse uuritava kemikaaliga mõõdetud väärtuse võrdlemisel negatiivse kontrolli fluorestseiini lekke väärtusega (negatiivse kontrolliga töödeldud rakkude konfluentse monokihiga proovi näit) ja maksimumlekke kontrolli väärtusega (rakkudeta vahemembraani kaudu lekkinud fluorestseiini näit).
Furthermore, highly cytotoxic test chemicals cannot always be reliably assessed.
Maksimumlekke fluorestsentsi intensiivsuse keskväärtus = x 0 % lekke (negatiivne kontroll) fluorestsentsi intensiivsuse keskväärtus = y 100 % lekke keskväärtus saadakse, kui 0 % lekke keskväärtus lahutatakse maksimumlekke keskväärtusest: x – y = z Iga fikseeritud annuse kohta arvutatakse lekke protsent, lahutades 0 % lekke väärtuse kolme paralleelproovi fluorestsentsi intensiivsuse näitude keskväärtusest (m) ning jagades tulemuse 100 % lekke väärtusega: % FL = [(m – y) / z] × 100 %, kus: M konkreetse kontsentratsiooni puhul kolmest paralleelproovist mõõdetud fluorestsentsi intensiivsuste keskväärtus % FL fluorestseiini protsent, mis lekib läbi rakukihi
Finally, test chemicals that interfere with the luciferase enzyme can confound the activity of luciferase in cell-based assays causing either apparent inhibition or increased luminescence (22).
Järgmist võrrandit kasutatakse 20 % FL leket põhjustava kontsentratsiooni arvutamiseks:
For example, phytoestrogen concentrations higher than 1 μM were reported to interfere with the luminescence signals in other luciferase-based reporter gene assays due to over-activation of the luciferase reporter gene (23).
FLD = [(A–B) / (C–B)] x (MC –MB) + MB
As a consequence, luciferase expression obtained at high concentrations of phytoestrogens or similar chemicals suspected of producing phytoestrogen-like over-activation of the luciferase reporter gene needs to be examined carefully (23).
Siin:
In cases where evidence can be demonstrated on the non-applicability of the test method to other specific categories of test chemicals, the test method should not be used for those specific categories.
D inhibitsiooni %
In addition to supporting discrimination between skin sensitisers and non-sensitisers, the KeratinoSensTM assay also provides concentration-response information that may potentially contribute to the assessment of sensitising potency when used in integrated approaches such as IATA (19).
A kahjustuse % (20 % fluorestseiini leket)
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.
B fluorestseiini lekke % < A
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.
C fluorestseiini lekke % > A
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).
C kontsentratsioon (mg/ml)
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).
B kontsentratsioon (mg/ml)
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.
läviväärtus silmi söövitavate / tugevalt ärritavate kemikaalide prognoosimiseks on esitatud allpool:
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)).
C&L: klassifitseerimine ja märgistamine
For this purpose, the maximal fold induction of the luciferase activity over solvent (negative) control (Imax) is determined.
ÜRO GHSi C&L
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 kategooria
Finally, parallel cytotoxicity measurements should be conducted to assess whether luciferase activity induction levels occur at sub-cytotoxic concentrations.
FL-katsemeetodit soovitatakse ainult vesilahustuvate silmi söövitavate ja tugevalt ärritavate (ÜRO GHSi 1. kategooria, ELi CLP-määruse 1. kategooria, US EPA I kategooria) kindlaks tegemiseks (vt punktid 1 ja 10).
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.
Selleks, et määrata vesilahustuv kemikaal (aine või segu) (3, 6, 7) rasket silmakahjustust tekitavaks (ÜRO GHSi / ELi CLP-määruse 1. kategooria) või silmi söövitavaks või tugevalt ärritavaks (US EPA I kategooria), peab uuritava kemikaaliga tekitatud FL20 väärtus olema ≤ 100 mg/ml.
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.
Tulemuste aktsepteerimine
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.
Fluorestseiini maksimumlekke keskväärtus (x) peab olema suurem kui 4000 (vt punkt 31), 0 % lekke keskväärtus (y) peab võrduma 300-ga või olema sellest väiksem ning 100 % lekke keskväärtus (z) peab jääma vahemikku 3700–6000.
Currently, the only method covered by this test method is the scientifically valid KeratinoSensTM assay (9) (12) (13) (14).
Katse loetakse nõuetekohaseks, kui positiivne kontroll tekitab rakukihi 20 %–40 % kahjustuse (mõõdetud fluorestseiini lekke protsendina).
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).
Andmed
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.
Iga katseseeria kohta tuleb tabelina esitada konkreetsete paralleelproovidega kannude andmed (nt fluorestsentsi intensiivsuse väärtused ja arvutatud fluorestseiini lekke protsent iga uuritava kemikaali kohta, sealhulgas klassifikatsioon).
The following paragraphs provide with a description of the main components and procedures of the ARE-Nrf2 luciferase test method.
Peale selle tuleb esitada iga katseseeria kohta konkreetsete paralleelsete mõõtetulemuste keskväärtused ± SD.
Preparation of the keratinocyte cultures
kemikaali CASi number, kui teada;
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).
aine või segu puhtus ja koostis (massiprotsendina/-protsentidena), selle teabe kättesaadavas ulatuses;
Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
uuringu läbiviimise seisukohast olulised füüsikalis-keemilised omadused (nt füüsikaline olek, lenduvus, pH, stabiilsus, lahustuvus vees, keemiliste ainete klass, kui teada);
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).
uuritava kemikaali / kontrolli kemikaali töötlemine enne katset, kui asjakohane (nt soojendamine, peenestamine);
For testing, cells should be 80-90 % confluent, and care should be taken to ensure that cells are never grown to full confluence.
säilitamistingimused.
One day prior to testing cells are harvested, and distributed into 96-well plates (10000 cells/well in the case of KeratinoSensTM).
Kasutatud meetodi ja metoodika põhjendus
Attention should be paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells.
Peab hõlmama katsemeetodi kohaldamisvaldkonna ja piirangutega seotud kaalutlusi.
If this is not the case, this step may give raise to high well-to-well variability.
Kasutatud rakusüsteemi kirjeldus, sealhulgas autentsussertifikaat ja mükoplasma staatus rakuliinis;
For each repetition, three replicates are used for the luciferase activity measurements, and one parallel replicate used for the cell viability assay.
kasutatud katse-eeskirja üksikasjad;
Preparation of the test chemical and control substances
uuritava kemikaali kasutatud kontsentratsioon(id);
The test chemical and control substances are prepared on the day of testing.
uuritava kemikaaliga kokkupuute kestus;
For the KeratinoSensTM assay, test chemicals are dissolved in dimethyl sulfoxide (DMSO) to the final desired concentration (e.g. 200 mM).
fluorestseiiniga inkubeerimise kestus;
The DMSO solutions can be considered self-sterilising, so that no sterile filtration is needed.
kasutatud hindamiskriteeriumide kirjeldus;
Test chemical not soluble in DMSO is dissolved in sterile water or culture medium, and the solutions sterilised by e.g. filtration.
viide mudeli varasemate katsete andmete kohta (st negatiivsed ja positiivsed kontrollid, võrdluskemikaalid, kui asjakohane);
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.
tõendid labori tehnilise pädevuse kohta.
In case solvents other than DMSO, water or the culture medium are used, sufficient scientific rationale should be provided.
Iga katseseeria konkreetse uuritava kemikaali ja kontrollide ning paralleelmõõtmiste tulemused tabelina (sealhulgas konkreetsed mõõtmistulemused, keskväärtused ja SD väärtused);
Based on the stock DMSO solutions of the test chemical, serial dilutions are made using DMSO to obtain 12 master concentrations of the chemical to be tested (from 0,098 to 200 mM in the KeratinoSensTM assay).
tuletatud klassifikatsioon(id) viitega prognoosimudelile ja/või kasutatud otsustamiskriteeriumidele;
For a test chemical not soluble in DMSO, the dilutions to obtain the master concentrations are made using sterile water or sterile culture medium.
Peab sisaldama kaalutlusi, mis on seotud ebaselge tulemuse (punkt 35: FL20 > 100 mg/ml) ja täiendavate katsete tegemisega.
Independent of the solvent used, the master concentrations, are then further diluted 25 fold into culture medium containing serum, and finally used for treatment with a further 4 fold dilution factor so that the final concentrations of the tested chemical range from 0,98 to 2000 μM in the KeratinoSensTM assay.
Järeldused
Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
Kättesaadav aadressil: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html]
The negative (solvent) control used in the KeratinoSensTM assay is DMSO (CAS No. 67-68-5, ≥ 99 % purity), for which six wells per plate are prepared.
Käesoleva lisa peatükk B.5 „Äge mürgisus: silmade ärritus/söövitus“.
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.
Kättesaadav aadressil: [http://ecvam-dbalm.jrc.ec.europa.eu
For a test chemical not soluble in DMSO, for which the dilutions were made in water, the DMSO level in all wells of the final test solution must be adjusted to 1 % as for the other test chemicals and control substances.
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.
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.
JOONIS.
Other suitable positive controls, preferentially providing EC1,5 values in the mid-range, may be used if historical data are available to derive comparable run acceptance criteria.
VAHEMEMBRAANIL KASVATATUD MCDK RAKKUDE KASUTAMINE FLUORESTSEIINI LEKKE KATSES
Application of the test chemical and control substances
Poolläbilaskval vahemembraanil kasvatatakse MCDK-rakkude konfluentne kiht.
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).
Vahemembraan pannakse 24-kannulise plaadi kannu.
In case of discordant results between the two independent repetitions, a third repetition containing three replicates should be performed (i.e. n = 9).
Joonise allikas: Wilkinson, P.J.
Each independent repetition is performed on a different day with fresh stock solution of test chemicals and independently harvested cells.
ELi CLP-määrus (määrus (EÜ) nr 1272/2008, mis käsitleb ainete ja segude klassifitseerimist, märgistamist ja pakendamist) selle määrusega rakendatakse Euroopa Liidus (ELis) kemikaalide (ainete ja segude) klassifitseerimise ja märgistamise ÜRO süsteem GHS.
Cells may come from the same passage however.
EPA I kategooria
After seeding as described in paragraph 20, cells are grown for 24 hours in the 96-wells microtiter plates.
väärtust saab hinnata, kui määrata uuritava kemikaali kontsentratsioon, millel kemikaal põhjustab fluorestseiini lekke 20 % läbi rakkude monokihi.
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.
Fluorestseiini leke
At least one well per plate should be left empty (no cells and no treatment) to assess background values.
GHSi 1. kategooria
The treated plates are then incubated for about 48 hours at 37 ± 1 °C in the presence of 5 % CO2 in the KeratinoSensTM assay.
kemikaal, mis põhjustab silma pöördumatut koekahjustust; b) kemikaalid, mis klassifitseeritakse ÜRO GHSi 1. kategooriasse; ELi CLP-määruse 1. kategooriasse; või US EPA silmi ärritava toimega kemikaalide I kategooriasse.
Luciferase activity measurements
Spetsiifilisus
Three factors are critical to ensure appropriate luminescence readings:
kõikide negatiivsete / inaktiivsete kemikaalide osakaal, mis katsemeetodi alusel klassifitseeritakse õigesti.
the choice of a sensitive luminometer,
Sellega mõõdetakse katsemeetodi täpsust, kui saadud tulemusi saab kategoriseerida, ning see on oluline aspekt katsemeetodi asjakohasuse hindamisel.
the use of a plate format with sufficient height to avoid light-cross-contamination; and
Tundlikkus
the use of a luciferase substrate with sufficient light output to ensure sufficient sensitivity and low variability.
kõikide positiivsete / toimet avaldavate kemikaalide osakaal, mis katsemeetodi alusel klassifitseeritakse õigesti.
Prior to testing, a control experiment setup as described in Appendix 3 should be carried out to ensure that these three points are met.
Seda terminit kasutatakse ka kordustäpsuse tähenduses, mis näitab, kui sageli annab katsemeetod õige tulemuse.
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.
Seda hinnatakse laborisisese ja laboritevahelise korratavuse ja laborisisese korduvuse arvutamisega.
Plates with the cell lysate are then placed in the luminometer for reading which in the KeratinoSensTM assay is programmed to: (i) add the luciferase substrate to each well (i.e. 50 μl), (ii) wait for 1 second, and (iii) integrate the luciferase activity for 2 seconds.
Valenegatiivsete tulemuste osakaal
In case alternative settings are used, e.g. depending on the model of luminometer used, these should be justified.
kõikide positiivsete kemikaalide osakaal, mis on katsemeetodiga ekslikult määratud negatiivseks.
Furthermore, a glow substrate may also be used provided that the quality control experiment of Appendix 3 is successfully fulfilled.
FL-KATSEMEETODI PÄDEVUSE KONTROLLI KEMIKAALID
Cytotoxicity Assessment
Enne käesoleva katsemeetodi tavakasutamist peab labor tõendama tehnilist pädevust ning määrama õigesti tabelis 1 soovitatud kemikaalidest 8 kemikaali kohta silmi söövitava toime klassifikatsiooni.
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.
Need kemikaalid on valitud nii, et nad esindaksid silma paikse söövituse / ärritava toime reaktsioonide vahemikku, mis põhineb in vivo küüliku silma katsega (katsejuhend nr 405, katsemeetod B.5 (5)) (st ÜRO GHSi 1. kategooria; 2A, 2B kategooria või klassifitseerimata).
The MTT medium is then removed and cells are lysed (e.g. by adding 10 % SDS solution to each well) overnight.
Võttes arvesse FL-katse valideeritud kasulikkust (st ainult silmi söövitavate / tugevalt ärritavate kemikaalide määramine), on klassifitseerimise seisukohast siiski pädevuse tõendamiseks olemas vaid kaks katse tulemust (söövitava / tugevalt ärritava toimega kemikaal või mittesöövitav/mitteärritav kemikaal).
After shaking, the absorption is measured at i.e. 600 nm with a photometer.
Kemikaalide valiku muud kriteeriumid olid kaubanduslik kättesaadavus, kvaliteetsete in vivo võrdlusandmete olemasolu ja FL-katsega saadud kvaliteetsete andmete olemasolu.