natgillin/translations-raw huggingface dataset:
Replication Index (RI)
Na podlagi skladnih rezultatov (reaktivnih ali nereaktivnih) med navidezno 100 mM raztopino in čisto kemikalijo je mogoče zanesljivo sklepati o rezultatu.
the proportion of cell division cycles completed in a treated culture, relative to the untreated control, during the exposure period and recovery (see Appendix 2 for formula).
Priprava pozitivnega kontrolnega vzorca, referenčnih kontrolnih vzorcev in koelucijskih kontrolnih vzorcev
mix of the S9 liver fraction and cofactors necessary for metabolic enzyme activity.
Za pozitivni kontrolni vzorec je treba uporabiti cimetov aldehid (št. CAS 104-55-2; s prehransko stopnjo čistosti ≥ 95 %) v koncentraciji 100 mM v acetonitrilu.
Untreated control
Uporabijo se lahko tudi drugi primerni pozitivni kontrolni vzorci, ki po možnosti zagotavljajo srednje vrednosti izgubljanja, če so na voljo podatki iz preteklih preskusov za izpeljavo primerljivih meril za sprejemljivost ponovitev.
When cytoB is used, evaluation of cytotoxicity should be based on the Cytokinesis-Block Proliferation Index (CBPI) or Replication Index (RI) (17) (69).
Poleg tega je treba referenčne kontrolne vzorce (tj. vzorce, ki vsebujejo le peptid, raztopljen v ustreznem topilu) vključiti v zaporedja ponovitev HPLC, ki se uporabijo za preverjanje ustreznosti sistema HPLC pred analizo (referenčni kontrolni vzorci A), stabilnosti referenčnih kontrolnih vzorcev v daljšem časovnem obdobju (referenčni kontrolni vzorci B) ter tega, da topilo, uporabljeno za raztopitev preskusne kemikalije, ne vpliva na odstotno vrednost izgube peptidov (referenčni kontrolni vzorci C) (glej Dodatek 3).
The CBPI indicates the average number of nuclei per cell, and may be used to calculate cell proliferation.
Z ustreznimi referenčnimi kontrolnimi vzorci za vsako kemikalijo se izračuna odstotna vrednost izgube peptidov za navedeno kemikalijo (glej odstavek 26).
The RI indicates the relative number of cell cycles per cell during the period of exposure to cytoB in treated cultures compared to control cultures and can be used to calculate the % cytostasis:
Poleg tega je treba v zaporedje ponovitev za vsako od analiziranih preskusnih kemikalij vključiti koelucijski kontrolni vzorec, sestavljen iz preskusne kemikalije same, da se zazna morebitna koelucija preskusne kemikalije s peptidom, sestavljenim iz lizina, ali peptidom, sestavljenim iz cisteina.
% Cytostasis = 100 – 100(CBPIT – 1) ÷ (CBPIC – 1)
Inkubacija preskusne kemikalije z raztopinama peptidov, sestavljenih iz cisteina in lizina
T test chemical treatment culture
Raztopino peptida, sestavljenega iz cisteina, je treba inkubirati v steklenih vialah za avtomatski vzorčevalnik s preskusno kemikalijo v razmerju 1: 10, raztopino peptida, sestavljenega iz lizina, pa v razmerju 1: 50.
C control culture
Če je takoj po tem, ko se v raztopino peptida doda raztopina preskusne kemikalije, vidna oborina, ki nastane zaradi slabe topnosti preskusne kemikalije v vodi, ne moremo biti prepričani o tem, koliko preskusne kemikalije je ostalo v raztopini za reagiranje s peptidom.
No. mononucleate cells
Zato se lahko v takem primeru še vedno uporabi pozitivni rezultat, negativni rezultat pa ni zanesljiv in ga je treba razlagati z ustrezno previdnostjo (glej tudi določbe v odstavku 11 za preskušanje kemikalij, ki do koncentracije 100 mM niso topne).
No. binucleate cells
Reakcijsko raztopino je treba pred izvedbo analize HPLC za 24 ur ± 2 uri pustiti v temi pri 25 ± 2,5 °C.
No. multinucleate cells
Vsako preskusno kemikalijo je treba analizirati v treh ponovitvah za oba peptida.
Total number of cells
Vzorce je treba pred analizo HPLC vizualno pregledati.
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Če se opazi oborina ali ločitev faz, se lahko vzorci centrifugirajo pri nizki hitrosti (100–400 x g), da se oborina izloči na dno viale kot previdnosti ukrep, saj lahko velike količine oborine zamašijo cevi ali kolone za HPLC.
Cytostasis = 100-RI
Če je oborina ali ločitev faz vidna po obdobju inkubacije, je vrednost izgubljanja peptidov morda podcenjena in v primeru negativnega rezultata ni mogoče dovolj zanesljivo sklepati o pomanjkanju reaktivnosti.
T treated cultures
Priprava standardne umeritvene krivulje za HPLC
C control cultures
Za peptide, sestavljene iz cisteina in lizina, je treba izdelati standardno umeritveno krivuljo.
Thus, an RI of 53 % means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53 % of this number divided in the treated culture, i.e. 47 % cytostasis.
Standardno raztopino peptida je treba pripraviti v 20- ali 25-odstotni raztopini acetonitrila in pufra, pri čemer se za peptid, sestavljen iz cisteina, uporabi fosfatni pufer (z vrednostjo pH 7,5), za peptid, sestavljen iz lizina, pa pufer amonijevega acetata (z vrednostjo pH 10,2).
When cytoB is not used, evaluation of cytotoxicity based on Relative Increase in Cell Counts (RICC) or on Relative Population Doubling (RPD) is recommended (69), as both take into account the proportion of the cell population which has divided.
Pripraviti je treba 6 raztopin za umerjanje, da se vanje zajame razpon od 0,534 do 0,0167 mM, pri čemer se uporabijo standardi za serije razredčitev osnovne raztopine peptida (0,667 mM).
Thus, a RICC, or a RPD of 53 % indicates 47 % cytotoxicity/cytostasis.
V standardno umeritveno krivuljo je treba vključiti tudi prazen vzorec s pufrom za redčenje.
By using a Proliferation Index (PI), cytotoxicity may be assessed via counting the number of clones consisting of 1 cell (cl1), 2 cells (cl2), 3 to 4 cells (cl4) and 5 to 8 cells (cl8).
Ustrezne umeritvene krivulje morajo imeti r2 > 0,99.
The PI has been used as a valuable and reliable cytotoxicity parameter also for cell lines cultured in vitro in the absence of cytoB (35) (36) (37) (38) and can be seen as a useful additional parameter.
Priprava in analiza HPLC
In any case, the number of cells before treatment should be the same for treated and negative control cultures.
Pred izvedbo analize je treba preveriti ustreznost sistema HPLC.
When using automated scoring systems, for instance, flow cytometry, laser scanning cytometry or image analysis, the number of cells in the formula can be substituted by the number of nuclei.
V sistem HPLC se vstavi ustrezna kolona.
This test method (TM) is equivalent to the OECD test guideline (TG) 442C (2015).
Vbrizgati je treba enake volumne standardne raztopine, vzorca in kontrolnega vzorca.
A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) (1) and Regulation (EC) No 1272/2008 of the European Parliament and Council on Classification, Labelling and Packaging of Substances and Mixtures (CLP) [12].
Kolono je treba med vbrizgavanji 7 minut ponovno uravnoteževati v začetnih pogojih.
This test method provides an in chemico procedure (Direct Peptide Reactivity Assay — DPRA) to be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with the UN GHS and CLP.
Če se uporabi druga kolona HPLC z reverzno fazo, je treba prilagoditi opisane parametre sistema, da se zagotovi ustrezno eluiranje in združevanje peptidov, sestavljenih iz cisteina in lizina, vključno z injekcijskim volumnom, ki se lahko spreminja glede na uporabljeni sistem (običajno v razponu 3 do 10 μl).
There is general agreement regarding the key biological events underlying skin sensitisation.
Pomembno je, da se v primeru uporabe drugega sistema HPLC prikaže, da je enakovreden opisanemu validiranemu sistemu (npr. s preskušanjem snovi za preverjanje usposobljenosti iz Dodatka 2).
The existing knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarised in the form of an Adverse Outcome Pathway (AOP) (2), from the molecular initiating event through the intermediate events to the adverse effect namely allergic contact dermatitis in humans or contact hypersensitivity in rodents.
Absorbanca se nadzoruje pri 220 nm.
Within the skin sensitisation AOP, the molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.
Če se uporablja detektor s serijo fotodiod, je treba zabeležiti tudi absorbanco pri 258 nm.
The assessment of skin sensitisation has typically involved the use of laboratory animals.
Opozoriti je treba, da imajo lahko nekatere pošiljke acetonitrila negativen učinek na stabilnost peptidov, kar je treba oceniti, ko se uporabi nova serija acetonitrila.
The classical methods based on guinea-pigs, the Magnusson Kligman Guinea Pig Maximisation Test (GMPT) and the Buehler Test (TM B.6 (3)), study both the induction and elicitation phases of skin sensitisation.
Kot kazalnik koelucije se lahko uporabi razmerje med površinama vrha pri 220 in 258.
A murine test, the Local Lymph Node Assay (LLNA, TM B.42 (4)) and its two non-radioactive modifications, LLNA: DA (TM B.50 (5)) and LLNA: BrdU-ELISA (TM B.51 (6)), which all assess the induction response exclusively, have also gained acceptance since they provide an advantage over the guinea pig tests in terms of animal welfare and an objective measurement of the induction phase of skin sensitisation.
Za vsak vzorec je razmerje v razponu 90 % < srednja [13] vrednost razmerja kontrolnih vzorcev < 100 % dober znak, da ni prišlo do koelucije.
More recently, mechanistically based in chemico and in vitro test methods have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.
Obstajajo preskusne kemikalije, ki lahko spodbujajo oksidacijo peptidov, sestavljenih iz cisteina.
However, combinations of non-animal methods (in silico, in chemico, in vitro) within Integrated Approaches to Testing and Assessment (IATA) will be needed to be able to fully substitute for the animal tests currently in use given the restricted AOP mechanistic coverage of each of the currently available non-animal test methods (2) (7).
Vrh dimeriziranih peptidov, sestavljenih iz cisteina, se lahko vizualno nadzoruje.
The DPRA is proposed to address the molecular initiating event of the skin sensitisation AOP, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine (8).
Če se pojavi dimerizacija, je treba to zabeležiti, saj se lahko odstotna vrednost izgube peptidov preceni, kar povzroči lažno pozitivne napovedi in/ali razvrstitev v višji razred reaktivnosti (glej odstavka 29 in 30).
Cysteine and lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers (9).
Analizo HPLC za peptide, sestavljene iz cisteina in lizina, se lahko izvede na isti (če sta na voljo dva sistema HPLC) ali na drug dan.
The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid (10) to be used as part of an IATA to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Če se analiza ne izvede na isti dan, je treba vsak dan sveže pripraviti vse raztopine preskusnih kemikalij za oba preskusa.
Examples on the use of DPRA data in combination with other information are reported in the literature (11) (12) (13) (14).
Pri analizi je treba meriti čas, da se zagotovi, da se vbrizgavanje prvega vzorca začne 22 do 26 ur po tem, ko je bila preskusna kemikalija zmešana z raztopino peptidov.
Definitions are provided in Appendix I.
Primer zaporedja analize HPLC je naveden v Dodatku 3.
INITIAL CONSIDERATIONS, APPLICABILITY AND LIMITATIONS
Ocenjevanje podatkov
The correlation of protein reactivity with skin sensitisation potential is well established (15) (16) (17).
Koncentracija peptidov, sestavljenih iz cisteina ali lizina, se v vsakem vzorcu fotometrično določi pri 220 nm tako, da se izmeri površina vrha (površina pod krivuljo – PPK) ustreznih vrhov in izračuna koncentracija peptidov z uporabo linearne umeritvene krivulje, izpeljane iz standardnih raztopin.
Nevertheless, since protein binding represents only one key event, albeit the molecular initiating event of the skin sensitisation AOP, protein reactivity information generated with testing and non-testing methods may not be sufficient on its own to conclude on the absence of skin sensitisation potential of chemicals.
Odstotna vrednost izgube peptidov se v vsakem vzorcu določi tako, da se izmeri površina vrha, ki se nato deli s srednjo vrednostjo površine vrha ustreznih referenčnih kontrolnih vzorcev C (glej Dodatek 3) v skladu s formulo, navedeno v nadaljevanju.
Therefore, data generated with this test method should be considered in the context of integrated approaches such as IATA, combining them with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods including read-across from chemical analogues.
Odstotna vrednost izgube peptidov
This test method can be used, in combination with other complementary information, to support the discrimination between skin sensitisers (i.e. UN GHS/CLP Category 1) and non-sensitisers in the context of IATA.
površina vrha peptida pri ponovnem vbrizganju
This test method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN GHS/CLP, nor to predict potency for safety assessment decisions.
srednja vrednost površine vrha peptida v ref.kontrolah C
However, depending on the regulatory framework, a positive result with the DPRA may be used on its own to classify a chemical into UN GHS/CLP category 1.
Merila za sprejemljivost
The DPRA test method proved to be transferable to laboratories experienced in high-performance liquid chromatography (HPLC) analysis.
Da se ponovitev šteje za veljavno, morajo biti izpolnjena naslednja merila:
The level of reproducibility in predictions that can be expected from the test method is in the order of 85 % within laboratories and 80 % between laboratories (10).
ustrezne umeritvene krivulje imajo r2 > 0,99;
Results generated in the validation study (18) and published studies (19) overall indicate that the accuracy of the DPRA in discriminating sensitisers (i.e. UN GHS/CLP Cat.
srednja odstotna vrednost izgube peptidov za tri ponovljene pozitivne kontrolne vzorce s cimetovim aldehidom se mora za peptide, sestavljene iz cisteina, gibati v razponu od 60,8 % do 100 %, za peptide, sestavljene iz lizina, pa v razponu od 40,2 % do 69,0 %, pri čemer je najvišji standardni odklon (SO) za ponovljene pozitivne kontrolne vzorce < 14,9 % za odstotek izgube cisteina in < 11,6 % za odstotek izgube lizina;
1) from non-sensitisers is 80 % (N=157) with a sensitivity of 80 % (88/109) and specificity of 77 % (37/48) when compared to LLNA results.
srednja vrednost koncentracije referenčnih kontrolnih vzorcev A mora znašati 0,50 ± 0,05 mM, koeficient variacije (KV) površine vrha peptidov za devet referenčnih kontrolnih vzorcev B in C v acetonitrilu pa < 15,0 %.
Furthermore when evaluating non-animal methods for skin sensitisation, it should be kept in mind that the LLNA test as well as other animal tests may not fully reflect the situation in the species of interest, i.e. humans.
najvišji standardni odklon za ponovljene vzorce preskusne kemikalije mora biti < 14,9 % za odstotek izgube cisteina in < 11,6 % za odstotek izgube lizina;
On the basis of the overall data available, the DPRA was shown to be applicable to test chemicals covering a variety of organic functional groups, reaction mechanisms, skin sensitisation potency (as determined in in vivo studies) and physico-chemical properties (8) (9) (10) (19).
srednja vrednost koncentracije peptidov treh referenčnih kontrolnih vzorcev C v ustreznem topilu mora biti 0,50 ± 0,05 mM.
Taken together, this information indicates the usefulness of the DPRA to contribute to the identification of skin sensitisation hazard.
Če ta merila niso izpolnjena, je treba podatke zavrniti in ponovitev za navedeno posebno preskusno kemikalijo izvesti še enkrat.
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 DPRA to the testing of substances and/or mixtures.
Napovedni model
This test method is not applicable for the testing of metal compounds since they are known to react with proteins with mechanisms other than covalent binding.
Za vsako preskusno kemikalijo se izračuna srednja odstotna vrednost izgube cisteina in lizina.
A test chemical should be soluble in an appropriate solvent at a final concentration of 100 mM (see paragraph 18).
Negativa izguba se pri izračunu srednje vrednosti šteje kot „0“.
However, test chemicals that are not soluble at this concentration may still be tested at lower soluble concentrations.
Če se uporablja napovedni model v razmerju 1: 10 za cistein in v razmerju 1: 50 za lizin iz preglednice 1, je treba v podporo razlikovanju med povzročitelji preobčutljivosti kože in snovmi, ki niso povzročitelji preobčutljivosti kože, v okviru IATA uporabiti prag v višini 6,38 % srednje izgube peptidov.
In such a case, a positive result could still be used to support the identification of the test chemical as a skin sensitiser but no firm conclusion on the lack of reactivity should be drawn from a negative result.
Uporaba napovednega modela za razvrstitev preskusne kemikalije v enega od razredov reaktivnosti (tj. nizka, zmerna in visoka reaktivnost) se lahko včasih izkaže za uporabno pri oblikovanju ocene moči v okviru IATA.
Limited information is currently available on the applicability of the DPRA to mixtures of known composition (18) (19).
Napovedni model v razmerju 1: 10 za cistein/ 1: 50 za lizin [14]
The DPRA is nevertheless considered to be technically applicable to the testing of multi-constituent substances and mixtures of known composition (see paragraph 18).
Srednja odstotna vrednost izgube cisteina in lizina
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.
Razred reaktivnosti
Such considerations are not needed when there is a regulatory requirement for testing of the mixture.
Napoved DPRA [15]
The current prediction model cannot be used for complex mixtures of unknown composition or for substances of unknown or variable composition, complex reaction products or biological materials (i.e. UVCB substances) due to the defined molar ratio of test chemical and peptide.
0 % ≤ srednja odstotna vrednost izgube ≤ 6,38 %
For this purpose a new prediction model based on a gravimetric approach will need to be developed.
brez reaktivnosti ali minimalna reaktivnost
In cases where evidence can be demonstrated on the non-applicability of the test method to other specific categories of chemicals, the test method should not be used for those specific categories of chemicals.
negativna
This test method is an in chemico method that does not encompass a metabolic system.
6,38 % < srednja odstotna vrednost izgube ≤ 22,62 %
Chemicals that require enzymatic bioactivation to exert their skin sensitisation potential (i.e. pro-haptens) cannot be detected by the test method.
nizka reaktivnost
Chemicals that become sensitisers after abiotic transformation (i.e. pre-haptens) are reported to be in some cases correctly detected by the test method (18).
pozitivna
In the light of the above, negative results obtained with the test method should be interpreted in the context of the stated limitations and in the connection with other information sources within the framework of an IATA.
22,62 % < srednja odstotna vrednost izgube ≤ 42,47 %
Test chemicals that do not covalently bind to the peptide but promote its oxidation (i.e. cysteine dimerisation) could lead to a potential over estimation of peptide depletion, resulting in possible false positive predictions and/or assignement to a higher reactivity class (see paragraphs 29 and 30).
zmerna reaktivnost
As described, the DPRA supports the discrimination between skin sensitisers and non-sensitisers.
42,47 % < srednja odstotna vrednost izgube ≤ 100 %
However, it may also potentially contribute to the assessment of sensitising potency (11) when used in integrated approaches such as IATA.
visoka reaktivnost
However further work, preferably based on human data, is required to determine how DPRA results may possibly inform potency assessment.
Lahko se pojavijo primeri, ko preskusna kemikalija (snov ali ena ali več sestavin snovi z več sestavinami ali zmesi) pri 220 nm močno absorbira in ima isti retencijski čas kot peptidi (koelucija).
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test chemical at 25 ± 2,5 °C.
Koelucija se lahko odpravi z manjšo prilagoditvijo sistema HPLC, da se nadalje loči elucijski čas preskusne kemikalije in peptidov.
The synthetic peptides contain phenylalanine to aid in the detection.
Če se poskuša koelucija odpraviti z uporabo drugega sistema HPLC, je treba prikazati, da je enakovreden validiranemu sistemu (npr. s preskušanjem snovi za preverjanje usposobljenosti iz Dodatka 2).
Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.
Ko pride do koelucije, vrha peptidov ni mogoče vključiti in izračun odstotne vrednosti izgube peptidov ni mogoč.
Cysteine- and lysine peptide percent depletion values are then calculated and used in a prediction model (see paragraph 29) which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
Če pride do koelucije takih preskusnih kemikalij s peptidi, sestavljenimi iz cisteina in lizina, je treba poročati, da je analiza „pomanjkljiva“.
Prior to routine use of the method described in this test method, laboratories should demonstrate technical proficiency, using the ten proficiency substances listed in Appendix 2.
Če pride do koelucije le s peptidi, sestavljenimi iz lizina, se lahko uporabi napovedni model v razmerju 1: 10 za cistein, o katerem se poroča v preglednici 2.
This test method is based on the DPRA DB-ALM protocol no 154 (20) which represents the protocol used for the EURL ECVAM-coordinated validation study.
Napovedni model v razmerju 1: 10 za cistein [16]
It is recommended that this protocol is used when implementing and using the method in the laboratory.
Odstotna vrednost izgube cisteina
The following is a description of the main components and procedures for the DPRA.
Razred reaktivnosti
If an alternative HPLC set-up is used, its equivalence to the validated set-up described in the DB-ALM protocol should be demonstrated (e.g. by testing the proficiency substances in Appendix 2).
Napoved DPRA [17]
Preparation of the cysteine or lysine-containing peptides
0 % ≤ odstotna vrednost izgube cisteina ≤ 13,89 %
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 85 % and preferably in the range of 90-95 %, should be freshly prepared just before their incubation with the test chemical.
13,89 % < odstotna vrednost izgube cisteina ≤ 23,09 %
The final concentration of the cysteine peptide should be 0,667 mM in pH 7,5 phosphate buffer whereas the final concentration of the lysine peptide should be 0,667 mM in pH 10,2 ammonium acetate buffer.
23,09 % < odstotna vrednost izgube cisteina ≤ 98,24 %
The HPLC run sequence should be set up in order to keep the HPLC analysis time less than 30 hours.
98,24 % < odstotna vrednost izgube cisteina ≤ 100 %