natgillin/translations-raw huggingface dataset:
The KeratinoSensTM assay is more likely to under predict chemicals showing a low to moderate skin sensitisation potency (i.e. UN GHS/CLP subcategory 1B) than chemicals showing a high skin sensitisation potency (i.e. UN GHS/CLP subcategory 1A) (13) (14).
odčitek povprečne absorbance MTT v jamicah, ki vsebujejo celice in kontrolo s topilom (negativno kontrolo).
Taken together, this information indicates the usefulness of the KeratinoSensTM assay to contribute to the identification of skin sensitisation hazard.
Vrednosti IC50 in IC30 se izračunata z linearno interpolacijo v skladu z enačbo 4, skupni vrednosti IC50 in IC30 pa se izračunata kot geometrijska sredina posameznih ponovitev.
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.
Enačba 4:
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.
X zmanjšanje koncentracije, ki jo je treba izračunati, v % (50 oz. 30 za IC50 oz. IC30);
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.
najnižja koncentracija v μM z zmanjšanjem viabilnosti > x %;
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).
najvišja koncentracija v μM z zmanjšanjem viabilnosti < x %;
Mainly mono-constituent substances were tested, although a limited amount of data also exist on the testing of mixtures (20).
viabilnost v % pri najnižji koncentraciji z zmanjšanjem viabilnosti > x %;
The test method is nevertheless technically applicable to the testing of multi-constituent substances and mixtures.
viabilnost v % pri najvišji koncentraciji z zmanjšanjem viabilnosti < x %.
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.
Pri vsaki koncentraciji, pri kateri se pokaže razmerje indukcije luciferazne dejavnosti > 1,5, se izračuna statistična značilnost (npr. z dvodelnim Studentovim t-testom), pri čemer se primerjajo vrednosti luminiscence za tri ponovljene vzorce z vrednostmi luminiscence v jamicah s kontrolo s topilom (negativna kontrola), da se določi, ali je indukcija luciferazne dejavnosti statistično značilna (p < 0,05).
Moreover, when testing multi-constituent substances or mixtures, consideration should be given to possible interference of cytotoxic constituents with the observed responses.
Najnižja koncentracija z razmerjem indukcije luciferazne dejavnosti > 1,5 je vrednost, ki določa vrednost EC1,5.
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).
V vsakem primeru se preveri, ali je ta vrednost nižja od vrednosti IC30, kar pomeni manj kot 30-odstotno zmanjšanje viabilnosti celic pri koncentraciji, s katero se določa EC1,5.
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.
Priporočeno je, da se podatki vizualno pregledajo z grafi.
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).
Če ni opaziti jasne krivulje odziva na odmerek ali če je krivulja odziva na odmerek bifazna (kar pomeni, da dvakrat prečka mejno vrednost 1,5), je treba poskus ponoviti, da se preveri, ali je to specifično za preskusno kemikalijo ali posledica poskusnega artefakta.
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).
Če je bifazni odziv mogoče ponoviti v neodvisnem poskusu, je treba poročati o nižji vrednosti EC1,5 (koncentracija, pri kateri je mejna vrednost 1,5 presežena prvič).
For substances having a LogP falling between 5 and 7, only limited information is available.
V redkih primerih, ko je opaziti statistično neznačilno razmerje indukcije nad 1,5, ki ji sledi višja koncentracija s statistično značilno indukcijo, se rezultati iz te ponovitve štejejo kot veljavni in pozitivni le, če je bilo za necitotoksično koncentracijo pridobljeno statistično značilno razmerje indukcije nad pragovno vrednostjo 1,5.
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.
Pri preskusnih kemikalijah, pri katerih se že pri najnižji preskusni koncentraciji, ki je 0,98 μM, ustvari razmerje indukcije v višini 1,5 ali več, se vrednost EC1,5 < 0,98 določi na podlagi vizualnega pregleda krivulje odziva na odmerek.
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.
Pri uporabi preskusa KeratinoSensTM morajo biti izpolnjena naslednja merila za sprejemljivost.
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).
Indukcija luciferazne dejavnosti, pridobljena s pozitivno kontrolo, tj. cimetovim aldehidom, mora v vsaj eni od preskušenih koncentracij (od 4 do 64 μM) statistično značilno presegati pragovno vrednost v višini 1,5 (npr. z uporabo t-preskusa).
Furthermore, highly cytotoxic test chemicals cannot always be reliably assessed.
Vrednost EC1,5 mora biti znotraj dveh standardnih odklonov srednje vrednosti preskusnega laboratorija iz preteklih preskusov (tj. med 7 μM in 30 μM na podlagi validacijske zbirke podatkov), ki jo je treba redno posodabljati.
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).
Poleg tega se mora povprečna indukcija treh ponovljenih vzorcev s cimetovim aldehidom pri 64 μM gibati v razponu od 2 do 8.
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).
Če slednje merilo ni izpolnjeno, je treba pazljivo preveriti razmerje med odmerkom in odzivom cimetovega aldehida, preskusi pa so sprejemljivi le, če obstaja jasno razmerje med odmerkom in odzivom z naraščajočo indukcijo luciferazne dejavnosti pri večjih koncentracijah za pozitivno kontrolo.
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).
Povprečni koeficient variacije odčitkov luminiscence za negativno kontrolo (kontrolo s topilom) v DMSO ne sme presegati 20 % pri vsaki ponovitvi, ki je sestavljena iz 6 jamic, preskušenih v treh ponovitvah.
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.
Če je variabilnost višja, je treba rezultate zavreči.
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).
Razlaga rezultatov in napovedni model
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.
Napoved KeratinoSensTM se obravnava kot pozitivna, če so izpolnjeni vsi naslednji štirje pogoji v dveh od dveh ponovitev ali v enakih dveh od treh ponovitev, v nasprotnem primeru se napoved KeratinoSensTM obravnava kot negativna (slika 1):
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.
Imax je višja od (>) 1,5-kratne in statistično značilno drugačna kot pri kontrolnem vzorcu s topilom (negativni kontroli) (kot je ugotovljeno z dvostranskim, neparnim Studentovim t-testom);
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).
viabilnost celic je višja od (>) 70 % pri najnižji koncentraciji z indukcijo dejavnosti luciferaze, ki presega 1,5-kratno (tj. pri koncentraciji za določanje EC1,5);
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).
vrednost EC1.5 je manjša od (<) 1000 μM (ali < 200 μg/ml za preskusne kemikalije brez opredeljene molekulske mase);
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.
za indukcijo luciferaze obstaja očiten splošen odziv na odmerek (ali dvofazen odziv, kot je omenjeno v odstavku 33).
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)).
Če so v dani ponovitvi izpolnjeni vsi prvi trije pogoji, ni pa mogoče ugotoviti jasnega odziva na odmerek za indukcijo luciferaze, je treba rezultat te ponovitve obravnavati kot nedokončen in morda bodo potrebni nadaljnji preskusi (slika 1).
For this purpose, the maximal fold induction of the luciferase activity over solvent (negative) control (Imax) is determined.
Poleg tega je treba kot nedokončnega obravnavati tudi negativen rezultat, pridobljen s koncentracijami < 1000 μM (ali < 200 μg/ml za preskusne kemikalije brez opredeljene molekulske mase) (glej odstavek 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).
Slika 1
Finally, parallel cytotoxicity measurements should be conducted to assess whether luciferase activity induction levels occur at sub-cytotoxic concentrations.
Napovedni model, uporabljen v preskus KeratinoSensTM.
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.
Napoved KeratinoSensTM je treba obravnavati v okviru IATA ter v skladu z določbami iz odstavkov 9 in 11.
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.
V redkih primerih so lahko preskusne kemikalije, ki povzročijo dejavnost luciferaze zelo blizu citotoksičnih ravni, pozitivne v nekaterih ponovitvah na necitotoksičnih ravneh (tj. koncentracija za določanje EC1,5 je nižja od (<) IC30), v drugih ponovitvah pa samo na citotoksičnih ravneh (tj. koncentracija za določanje EC1,5 je višja od (>) IC30).
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.
Take preskusne kemikalije je treba ponovno preskusiti z ožjo analizo odziva na odmerek, pri čemer se uporabi nižji faktor razredčenja (npr. 1,33- ali Ö2 (= 1,41)-kratno razredčenje med jamicami), da se ugotovi, ali je do indukcije prišlo na citotoksičnih ravneh ali ne (9).
Currently, the only method covered by this test method is the scientifically valid KeratinoSensTM assay (9) (12) (13) (14).
fizični videz, topnost v vodi, topnost v DMSO, molekulska masa in dodatne pomembne fizikalno-kemijske lastnosti, če so na voljo;
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).
fizični videz, topnost v vodi, topnost v DMSO in dodatne pomembne fizikalno-kemijske lastnosti, če so na voljo;
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.
fizični videz, topnost v vodi, topnost v DMSO, molekulska masa in dodatne pomembne fizikalno-kemijske lastnosti, če so na voljo in če je ustrezno;
The following paragraphs provide with a description of the main components and procedures of the ARE-Nrf2 luciferase test method.
negativna kontrola (z vehiklom):
Preparation of the keratinocyte cultures
kemijski identifikacijski podatki, kot so ime po IUPAC ali CAS, številka CAS in/ali drugi identifikatorji;
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).
fizični videz, molekulska masa in dodatne pomembne fizikalno-kemijske lastnosti, če se uporabijo druge negativne kontrole/vehikli razen tistih, ki so navedeni pri tej preskusni metodi, in če so na voljo;
Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
utemeljitev izbire topila za vsako preskusno kemikalijo.
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).
Pogoji preskusne metode:
For testing, cells should be 80-90 % confluent, and care should be taken to ensure that cells are never grown to full confluence.
opis uporabljene preskusne metode;
One day prior to testing cells are harvested, and distributed into 96-well plates (10000 cells/well in the case of KeratinoSensTM).
uporabljena celična linija, pogoji njenega hranjenja in vir (npr. laboratorij, ki jo je zagotovil);
Attention should be paid to avoid sedimentation of the cells during seeding to ensure homogeneous cell number distribution across wells.
število pasaž in stopnja konfluence celic, uporabljenih za preskušanje;
If this is not the case, this step may give raise to high well-to-well variability.
metoda štetja celic, uporabljena za nasaditev pred preskušanjem, in ukrepi, sprejeti za zagotovitev homogene porazdelitve števila celic (glej tudi odstavek 20);
For each repetition, three replicates are used for the luciferase activity measurements, and one parallel replicate used for the cell viability assay.
uporabljeni luminometer (npr. model), vključno z nastavitvami instrumenta, uporabljenim substratom luciferaze in prikazom ustreznih meritev luminiscence, ki temeljijo na kontrolnem preskusu, opisanem v Dodatku 3;
Preparation of the test chemical and control substances
postopek, uporabljen za dokazovanje usposobljenosti laboratorija v zvezi z izvedbo preskusne metode (npr. s preskušanjem snovi za preverjanje usposobljenosti) ali za prikaz ponovljivosti izvedbe preskusne metode v daljšem časovnem obdobju.
The test chemical and control substances are prepared on the day of testing.
Preskusni postopek:
For the KeratinoSensTM assay, test chemicals are dissolved in dimethyl sulfoxide (DMSO) to the final desired concentration (e.g. 200 mM).
uporabljeno število ponovitev in ponovljenih vzorcev;
The DMSO solutions can be considered self-sterilising, so that no sterile filtration is needed.
uporabljene koncentracije preskusnih kemikalij, postopek dodajanja in čas izpostavljenosti (če je drugačen od priporočenega);
Test chemical not soluble in DMSO is dissolved in sterile water or culture medium, and the solutions sterilised by e.g. filtration.
Preglednice z vrednostmi Imax, EC1.5 in vrednostmi viabilnosti (tj. IC50, IC30), ki so bile pridobljene za preskusno kemikalijo in pozitivno kontrolo za vsako ponovitev, ter srednje vrednosti (Imax: povprečje; EC1.5 in vrednosti viabilnosti: geometrijska sredina) in standardni odklon (SD), izračunan na podlagi podatkov iz vseh posameznih ponovitev, ter indikacija ocene preskusne kemikalije glede na napovedni model;
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.
koeficient variacije, pridobljen z meritvami luminiscence za negativno kontrolo za vsak poskus;
In case solvents other than DMSO, water or the culture medium are used, sufficient scientific rationale should be provided.
graf, ki prikazuje krivulje odziva na odmerek za indukcijo dejavnosti luciferaze in viabilnost;
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).
opis kakršnih koli drugih pomembnih opažanj, če je ustrezno.
For a test chemical not soluble in DMSO, the dilutions to obtain the master concentrations are made using sterile water or sterile culture medium.
razprava o rezultatih, pridobljenih s preskusom KeratinoSensTM;
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.
obravnavanje rezultatov preskusnih metod v okviru IATA, če so na voljo druge pomembne informacije.
Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
Na voljo na: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_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.
OECD Environment, Health and Safety publications, Series on Testing and Assessment No.
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.
OECD, Pariz.
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.
Poglavje B.42 te priloge: Preobčutljivost kože: lokalna analiza bezgavk.
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.
Poglavje B.50 te priloge: Preobčutljivost kože: lokalna analiza bezgavk: DA.
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.
Poglavje B.51 te priloge: Preobčutljivost kože: lokalna analiza bezgavk: BrdU-ELISA.
Application of the test chemical and control substances
Recommendation on the KeratinoSensTM assay for skin sensitisation testing, 42 str.
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).
Na voljo na: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/recommendation-keratinosens-skin-sensitisation.
In case of discordant results between the two independent repetitions, a third repetition containing three replicates should be performed (i.e. n = 9).
DB-ALM (INVITTOX) (2013).
Each independent repetition is performed on a different day with fresh stock solution of test chemicals and independently harvested cells.
Protocol 155: KeratinoSensTM., 17 str.
Cells may come from the same passage however.
Na voljo na: http://ecvam-dbalm.jrc.ec.europa.eu/beta/index.cfm/methodsAndProtocols/index.
After seeding as described in paragraph 20, cells are grown for 24 hours in the 96-wells microtiter plates.
OECD Guidelines for Chemical Testing No.
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.
Ta izraz in izraz „skladnost“ se pogosto uporabljata kot sopomenki in pomenita delež pravilnih rezultatov preskusne metode (29).
At least one well per plate should be left empty (no cells and no treatment) to assess background values.
(imenuje se tudi EpRE oziroma element elektrofilnega odziva) je element odziva v zgornji promotorski regiji mnogih genov za zaščito celic in genov faze II.
The treated plates are then incubated for about 48 hours at 37 ± 1 °C in the presence of 5 % CO2 in the KeratinoSensTM assay.
Ko se aktivira z Nfr2, posreduje v transkripcijski indukciji teh genov.
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.
interpolirana koncentracija za 1,5-kratno indukcijo luciferaze.
Luciferase activity measurements
koncentracija, ki vpliva na zmanjšanje viabilnosti celic za 30 %.
Three factors are critical to ensure appropriate luminescence readings:
koncentracija, ki vpliva na zmanjšanje viabilnosti celic za 50 %.
the choice of a sensitive luminometer,
največji faktor indukcije luciferazne dejavnosti v primerjavi s kontrolnim vzorcem s topilom (negativno kontrolo), ki se meri za vse koncentracije preskusnih kemikalij.
the use of a plate format with sufficient height to avoid light-cross-contamination; and
„Kelch-like ECH-associated protein 1“ je senzorska beljakovina, ki lahko uravnava dejavnost Nrf2.