natgillin/translations-raw huggingface dataset:
For the HPLC set up used in the validation study and described in this test method, up to 26 analysis samples (which include the test chemical, the positive control and the appropriate number of solvent controls based on the number of individual solvents used in the test, each tested in triplicate), can be accommodated in a single HPLC run.
Obstajajo lahko tudi drugi primeri, pri katerih prihaja do prekrivanja retencijskega časa med preskusno kemikalijo in je kateri koli od peptidov nepopoln.
All of the replicates analysed in the same run should use the identical cysteine and lysine peptide stock solutions.
V takih primerih se lahko odstotne vrednosti izgube peptidov ocenijo in uporabijo v napovednem modelu, pri katerem je cistein v razmerju 1: 10, lizin pa v razmerju 1: 50, vendar preskusne kemikalije ni mogoče natančno razvrstiti v enega od razredov reaktivnosti.
It is recommended to prove individual peptide batches for proper solubility prior to their use.
Kadar je rezultat nedvoumen, za preskusno kemikalijo zadostuje ena sama analiza HPLC za peptide, sestavljene iz cisteina in lizina.
Preparation of the test chemical
Kadar pa so rezultati blizu mejne vrednosti, ki se uporablja za razlikovanje med pozitivnimi in negativnimi rezultati (tj. so rezultati mejni), je potrebno dodatno preskušanje.
Solubility of the test chemical in an appropriate solvent should be assessed before performing the assay following the solubilisation procedure described in the DPRA DB-ALM protocol (20).
V primerih, ko se srednja odstotna vrednost izgube pri napovednem modelu, pri katerem je cistein v razmerju 1: 10, lizin pa v razmerju 1: 50, giblje v razponu od 3 % do 10 % ali se odstotne vrednosti izgube cisteina pri modelu za napovedovanje, pri katerem je cistein v razmerju 1: 10, gibljejo v razponu od 9 % do 17 %, je treba razmisliti o drugi ponovitvi, v primeru neskladnih rezultatov med prvima dvema ponovitvama pa tudi o tretji.
An appropriate solvent will dissolve the test chemical completely.
V poročilo o preskusu se vključijo naslednji podatki:
Since in the DPRA the test chemical is incubated in large excess with either the cysteine or the lysine peptides, visual inspection of the forming of a clear solution is considered sufficient to ascertain that the test chemical (and all of its components in the case of testing a multi-constituent substance or a mixture) is dissolved.
Snov iz ene sestavine:
Suitable solvents are acetonitrile, water, 1:1 mixture water:acetonitrile, isopropanol, acetone or 1:1 mixture acetone:acetonitrile.
kemijski identifikacijski podatki, kot so ime po IUPAC ali CAS, številka CAS, oznaka po sistemu SMILES ali identifikator InChI, strukturna formula in/ali drugi identifikatorji;
Other solvents can be used as long as they do not impact on the stability of the peptide as monitored with reference controls C (i.e. samples constituted by the peptide alone dissolved in the appropriate solvent; see Appendix 3).
fizični videz, topnost v vodi, molekulska masa in dodatne pomembne fizikalno-kemijske lastnosti, če so na voljo;
As a last option if the test chemical is not soluble in any of these solvents attempts should be made to solubilise it in 300 μL of DMSO and dilute the resulting solution with 2700 μL of acetonitrile and if the test chemical is not soluble in this mixture attempts should be made to solubilise the same amount of test chemical in 1500 μL of DMSO and dilute the resulting solution with 1500 μL of acetonitrile.
čistost, kemijska identiteta nečistot, kot je ustrezno in praktično izvedljivo, itd.;
The test chemical should be pre-weighed into glass vials and dissolved immediately before testing in an appropriate solvent to prepare a 100 mM solution.
obdelava pred preskušanjem, če je ustrezno (npr. segrevanje, mletje);
For mixtures and multi-constituent substances of known composition, a single purity should be determined by the sum of the proportion of its constituents (excluding water), and a single apparent molecular weight should be determined by considering the individual molecular weights of each component in the mixture (excluding water) and their individual proportions.
preskušene koncentracije;
The resulting purity and apparent molecular weight should then be used to calculate the weight of test chemical necessary to prepare a 100 mM solution.
pogoji shranjevanja in stabilnost, če so na voljo.
For polymers for which a predominant molecular weight cannot be determined, the molecular weight of the monomer (or the apparent molecular weight of the various monomers constituting the polymer) may be considered to prepare a 100 mM solution.
snov z več sestavinami, UVCB in zmesi:
However, when testing mixtures, multi-constituent substances or polymers of known composition, it should be considered to also test the neat chemical.
čim obsežnejša opredelitev lastnosti, na primer s kemijsko identiteto (glej zgoraj), čistostjo, kvantitativnim pojavljanjem in ustreznimi fizikalno-kemijskimi lastnostmi (glej zgoraj) sestavin, če so na voljo;
For liquids, the neat chemical should be tested as such without any prior dilution by incubating it at 1:10 and 1:50 molar ratio with the cysteine and lysine peptides, respectively.
fizični videz, topnost v vodi in dodatne pomembne fizikalno-kemijske lastnosti, če so na voljo;
For solids, the test chemical should be dissolved to its maximum soluble concentration in the same solvent used to prepare the apparent 100 mM solution.
molekulska masa ali navidezna molekulska masa v primeru zmesi/polimerov z znano sestavo ali drugi podatki, pomembni za izvedbo študije;
It should then be tested as such without any further dilution by incubating it at 1:10 and 1:50 ratio with the cysteine and lysine peptides, respectively.
pogoji shranjevanja in stabilnost, če so na voljo;
Concordant results (reactive or non-reactive) between the apparent 100 mM solution and the neat chemical should allow for a firm conclusion on the result.
sklicevanje na rezultate pozitivnih kontrol iz preteklih preskusov, ki kažejo ustrezna merila za sprejemljivost ponovitev, če je to ustrezno;
Preparation of the positive control, reference controls and coelution controls
uporabljeno topilo/vehikel in razmerja sestavin, če so na voljo;
Cinnamic aldehyde (CAS 104-55-2; ≥ 95 % food-grade purity) should be used as positive control (PC) at a concentration of 100 mM in acetonitrile.
kemijski identifikacijski podatki, kot so ime po IUPAC ali CAS, številka CAS in/ali drugi identifikatorji;
Other suitable positive controls preferentially providing mid-range depletion values may be used if historical data are available to derive comparable run acceptance criteria.
fizični videz, molekulska masa in dodatne pomembne fizikalno-kemijske lastnosti, če se uporabijo druga topila/vehikli razen tistih, ki so navedeni pri tej preskusni metodi, in če so na voljo;
In addition, reference controls (i.e. samples containing only the peptide dissolved in the appropriate solvent) should also be included in the HPLC run sequence and these are used to verify the HPLC system suitability prior to the analysis (reference controls A), the stability of the reference controls over time (reference controls B) and to verify that the solvent used to dissolve the test chemical does not impact the percent peptide depletion (reference controls C) (see Appendix 3).
utemeljitev izbire topila za vsako preskusno kemikalijo;
The appropriate reference control for each chemical is used to calculate the percent peptide depletion for that chemical (see paragraph 26).
za acetonitril – rezultati testa učinka na stabilnost peptida.
Incubation of the test chemical with the cysteine and lysine peptide solutions
opredelitev značilnosti raztopine peptidov (dobavitelj, serija, natančna teža peptidov, volumen, ki se doda za osnovno raztopino);
Cysteine and lysine peptide solutions should be incubated in glass autosampler vials with the test chemical at 1:10 and 1:50 ratio respectively.
opredelitev značilnosti pozitivne kontrole (natančna teža snovi za pozitivno kontrolo, volumen, ki se doda za preskusno raztopino);
If a precipitate is observed immediately upon addition of the test chemical solution to the peptide solution, due to low aqueous solubility of the test chemical, in this case one cannot be sure how much test chemical remained in the solution to react with the peptide.
opredelitev značilnosti raztopin s preskusno kemikalijo (natančna teža preskusne kemikalije, volumen, ki se doda za preskusno raztopino).
Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and should be interpreted with due care (see also provisions in paragraph 11 for the testing of chemicals not soluble up to a concentration of 100 mM).
Nastavitve in analiza instrumenta za tekočinsko kromatografijo visoke ločljivosti (HPLC)
The reaction solution should be left in the dark at 25 ± 2,5 °C for 24 ± 2 hours before running the HPLC analysis.
vrsta instrumenta za HPLC, kolone za HPLC in predkolone, detektor, avtomatski vzorčevalnik;
Each test chemical should be analysed in triplicate for both peptides.
parametri, ki so pomembni za analizo HPLC, kot so temperatura kolone, injekcijski volumen, pretok in gradient.
Samples have to be visually inspected prior to HPLC analysis.
Ustreznost sistema
If a precipitate or phase separation is observed, samples may be centrifuged at low speed (100-400 xg) to force precipitate to the bottom of the vial as a precaution since large amounts of precipitate may clog the HPLC tubing or columns.
površina vrha peptida pri 220 nm v vsaki standardni raztopini in ponovljenem referenčnem kontrolnem vzorcu A;
If a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result.
grafično prikazana linearna umeritvena krivulja in vrednost r2, o kateri se je poročalo;
Preparation of the HPLC standard calibration curve
koncentracija peptida v vsakem ponovljenem referenčnem kontrolnem vzorcu A;
A standard calibration curve should be generated for both the cysteine and the lysine peptides.
srednja vrednost koncentracije (v mM) peptida v treh referenčnih kontrolnih vzorcih A, SD in CV;
Peptide standards should be prepared in a solution of 20 % or 25 % acetonitrile:buffer using phosphate buffer (pH 7,5) for the cysteine peptide and ammonium acetate buffer (pH 10,2) for the lysine peptide.
koncentracija peptida v referenčnih kontrolnih vzorcih A in C.
Using serial dilution standards of the peptide stock solution (0,667 mM), 6 calibration solutions should be prepared to cover the range from 0,534 to 0,0167 mM.
Potek analize:
A blank of the dilution buffer should also be included in the standard calibration curve.
za referenčne kontrolne vzorce:
Suitable calibration curves should have an r2 > 0,99.
površina vrha peptida pri 220 nm v vsakem ponovljenem kontrolnem vzorcu B in C;
HPLC preparation and analysis
srednja vrednost površine vrha peptida pri 220 nm v devetih referenčnih kontrolnih vzorcih B in C v acetonitrilu, SD in CV (za stabilnost referenčnih kontrolnih vzorcev med trajanjem analize);
The suitability of the HPLC system should be verified before conducting the analysis.
za vsako uporabljeno topilo: srednja vrednost površine vrha peptida pri 220 nm v treh ustreznih referenčnih kontrolnih vzorcih C (za izračun odstotne vrednosti izgube peptidov);
Peptide depletion is monitored by HPLC coupled with an UV detector (photodiode array detector or fixed wavelength absorbance detector with 220 nm signal).
za vsako uporabljeno topilo: koncentracija peptida (v mM) v treh ustreznih referenčnih kontrolnih vzorcih C;
The appropriate column is installed in the HPLC system.
za vsako uporabljeno topilo: srednja vrednost koncentracije peptida (v mM) v treh ustreznih referenčnih kontrolnih vzorcih C, SD in CV;
The HPLC set-up described in the validated protocol uses a Zorbax SB-C-18 2,1 mm × 100 mm × 3,5 micron as preferred column.
za pozitivni kontrolni vzorec:
With this reversed-phase HPLC column, the entire system should be equilibrated at 30 °C with 50 % phase A (0,1 % (v/v) trifluoroacetic acid in water) and 50 % phase B (0,085 % (v/v) trifluoroacetic acid in acetonitrile) for at least 2 hours before running.
površina vrha peptida pri 220 nm za vsak ponovljeni vzorec;
The HPLC analysis should be performed using a flow rate of 0,35 ml/min and a linear gradient from 10 % to 25 % acetonitrile over 10 minutes, followed by a rapid increase to 90 % acetonitrile to remove other materials.
odstotna vrednost izgube peptidov za vsak ponovljeni vzorec;
Equal volumes of each standard, sample and control should be injected.
srednja odstotna vrednost izgube peptidov za tri ponovljene vzorce, SD in CV;
The column should be re-equilibrated under initial conditions for 7 minutes between injections.
za vsako preskusno kemikalijo:
If a different reversed-phase HPLC column is used, the set-up parameters described above may need to be adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides, including the injection volume, which may vary according to the system used (typically in the range from 3-10 μl).
prisotnost koelucije;
Importantly, if an alternative HPLC set-up is used, its equivalence to the validated set-up described above should be demonstrated (e.g. by testing the proficiency substances in Appendix 2).
opis kakršnih koli drugih pomembnih opažanj, če je ustrezno;
Absorbance is monitored at 220 nm.
srednja odstotna vrednost izgube peptidov v treh ponovljenih vzorcih, SD in CV;
If a photodiode array detector is used, absorbance at 258 nm should also be recorded.
srednje odstotne vrednosti izgube cisteina in lizina;
It should be noted that some supplies of acetonitrile could have a negative impact on peptide stability and this has to be assessed when a new batch of acetonitrile is used.
uporabljeni napovedni model in napoved DPRA.
The ratio of the 220 peak area and the 258 peak area can be used as an indicator of co-elution.
Preskušanje usposobljenosti:
For each sample a ratio in the range of 90 % < mean [13] area ratio of control samples < 100 % would give a good indication that co-elution has not occurred.
če je ustrezno, 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.
There may be test chemicals which could promote the oxidation of the cysteine peptide.
Razprava o rezultatih
The peak of the dimerised cysteine peptide may be visually monitored.
razprava o rezultatih, pridobljenih s preskusno metodo DPRA;
If dimerisation appears to have occurred, this should be noted as percent peptide depletion may be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class (see paragraphs 29 and 30).
razprava o rezultatih preskusne metode v okviru IATA, če so na voljo drugi pomembni podatki.
HPLC analysis for the cysteine and lysine peptides can be performed concurrently (if two HPLC systems are available) or on separate days.
Na voljo na: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html.
If analysis is conducted on separate days then all test chemical solutions should be freshly prepared for both assays on each day.
Part 1: Scientific Evidence.
The analysis should be timed to assure that the injection of the first sample starts 22 to 26 hours after the test chemical was mixed with the peptide solution.
Series on Testing and Assessment št. 168, OECD, Pariz.
For the HPLC set up used in the validation study and described in this test method, up to 26 analysis samples can be accommodated in a single HPLC run (see also paragraph 17).
Poglavje B.6 te priloge: Senzibilizacija kože.
An example of HPLC analysis sequence is provided in Appendix 3.
Poglavje B.42 te priloge: Lokalna analiza bezgavk.
Data evaluation
Poglavje B.50 te priloge: Preobčutljivost kože: lokalna analiza bezgavk: DA.
The concentration of cysteine or lysine peptide is photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
Poglavje B.51 te priloge: Preobčutljivost kože: lokalna analiza bezgavk: BrdU-ELISA.
The percent peptide depletion is determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C (see Appendix 3) according to the formula described below.
Na voljo na: https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations/eurl-ecvam-recommendation-on-the-direct-peptide-reactivity-assay-dpra.
Percent peptide depletion
Na voljo na: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/eurl-ecvam-recommendation-on-the-direct-peptide-reactivity-assay-dpra.
Peptide peak area in replicate injection
Na voljo na: http://ecvam-dbalm.jrc.ec.europa.eu/.
ean peptide peak area in reference controls C
OECD Series on Testing and Assessment, št. 34.
Acceptance criteria
Organisation for Economic Cooperation and Development, Pariz, Francija.
The following criteria should be met for a run to be considered valid:
Na voljo na: www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidance/ucm070107.pdf – 138.
the standard calibration curve should have an r2 > 0,99,
Ta izraz in izraz „skladnost“ se pogosto uporabljata kot sopomenki in pomenita delež pravilnih rezultatov preskusne metode (21).
the mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60,8 % and 100 % for the cysteine peptide and between 40,2 % and 69,0 % for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14,9 % for the percent cysteine depletion and < 11,6 % for the percent lysine depletion, and
Potek neželenega izida (Adverse Outcome Pathway – AOP)
the mean peptide concentration of reference controls A should be 0,50 ± 0,05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be < 15,0 %.
zaporedje dogodkov od kemijske strukture ciljne kemikalije ali skupine podobnih kemikalij prek začetnega dogodka na molekulski ravni do preiskovanega rezultata in vivo (2).
If one or more of these criteria is not met the run should be repeated.
Umeritvena krivulja
The following criteria should be met for a test chemical's results to be considered valid:
razmerje med vrednostjo odziva med preskusom in analitsko koncentracijo (imenovana tudi standardna krivulja) znane snovi.
the maximum standard deviation for the test chemical replicates should be < 14,9 % for the percent cysteine depletion and < 11,6 % for the percent lysine depletion,
Koeficient variacije
the mean peptide concentration of the three reference controls C in the appropriate solvent should be 0,50 ± 0,05 mM.
merilo variabilnosti, ki se za skupino podatkov za ponovljene vzorce izračuna tako, da se standardni odklon deli s srednjo vrednostjo.
If these criteria are not met the data should be rejected and the run should be repeated for that specific test chemical.
Lahko se pomnoži s 100, da se izrazi v odstotkih.
Prediction model
IATA (Integrated Approach to Testing and Assessment = celostni pristop k testiranju in ocenjevanju)
The mean percent cysteine and percent lysine depletion value is calculated for each test chemical.
strukturiran pristop, ki se uporablja za ugotavljanje nevarnosti (potencial), opredeljevanje nevarnosti (moč) in/ali oceno varnosti (potencial/moč in izpostavljenost) kemikalije ali skupine kemikalij ter strateško zajame in oceni vse pomembne podatke, da se lahko sprejme regulativna odločitev v zvezi z morebitno nevarnostjo in/ali tveganjem in/ali potrebo po nadaljnjem, ciljno usmerjenem in s tem minimalnem preskušanju.
Negative depletion is considered as “0” when calculating the mean.
Začetni dogodek na molekulski ravni
By using the cysteine 1:10/lysine 1:50 prediction model shown in Table 1, the threshold of 6,38 % average peptide depletion should be used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.
kemijsko povzročena sprememba biološkega sistema na molekulski ravni, ki se v poteku neželenega izida opredeli kot začetni dogodek.
Application of the prediction model for assigning a test chemical to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.
zmes ali raztopina iz dveh ali več snovi, ki v njej ne reagirajo (1).
Cysteine 1:10/lysine 1:50 prediction model [14]
snov, ki jo opredeljuje njena kvantitativna sestava in v kateri je vsaj 80 mas. % glavne sestavine.
Mean of cysteine and lysine % depletion
Snov z več sestavinami
Reactivity Class
snov, ki jo opredeljuje njena kvantitativna sestava in v kateri je več glavnih sestavin s koncentracijo ≥ 10 mas. % in < 80 mas. %.
DPRA Prediction [15]
Snov z več sestavinami je rezultat proizvodnega postopka.
0 % ≤ mean % depletion ≤ 6,38 %
Razlika med zmesjo in snovjo z več sestavinami je v tem, da je zmes pridobljena z mešanjem dveh ali več snovi brez kemične reakcije.
No or minimal reactivity
Snov z več sestavinami je rezultat kemične reakcije.
Negative
ponovljen vzorec, ki vsebuje vse sestavine preskusnega sistema in se tretira s snovjo, za katero je znano, da povzroči pozitiven odziv.
6,38 % < mean % depletion ≤ 22,62 %
Referenčna kontrola
Low reactivity
netretiran vzorec, ki vsebuje vse komponente preskusnega sistema, vključno s topilom ali vehiklom, ki se obdela s kontrolnimi vzorci, tretiranimi s preskusno kemikalijo, in drugimi kontrolnimi vzorci, da se določi izhodiščni odziv za vzorce, tretirane s preskusno kemikalijo, raztopljeno v istem topilu ali vehiklu.
Positive
Ustreznost
22,62 % < mean % depletion ≤ 42,47 %
opis razmerja med preskusom in preiskovanim učinkom ter njegovega pomena in uporabnosti za določen namen.
Moderate reactivity
Pomeni stopnjo, do katere preskus pravilno izmeri ali napove preiskovani biološki učinek.
42,47 % < mean % depletion ≤ 100 %
Ustreznost upošteva tudi točnost (skladnost) preskusne metode (21).
High reactivity
Oceni se z izračunom obnovljivosti v enem ali več laboratorijih in interne laboratorijske ponovljivosti (21).
There might be cases where the test chemical (the substance or one or several of the components of a multi-constituent substance or a mixture) absorbs significantly at 220 nm and has the same retention time of the peptide (co-elution).
Obnovljivost
Co-elution may be resolved by slightly adjusting the HPLC set-up in order to further separate the elution time of the test chemical and the peptide.
ujemanje rezultatov, pridobljenih s preskušanjem iste kemikalije in uporabo istega preskusnega protokola (glej zanesljivost) (21).
If an alternative HPLC set-up is used to try to resolve co-elution, its equivalence to the validated set-up should be demonstrated (e.g. by testing the proficiency substances in Appendix 2).
delež vseh pozitivnih/aktivnih kemikalij, ki se s preskusno metodo pravilno razvrstijo.
When co-elution occurs the peak of the peptide cannot be integrated and the calculation of the percent peptide depletion is not possible.
Je merilo točnosti za preskusno metodo, s katero se pridobijo kategorični rezultati, in pomemben dejavnik pri ocenjevanju ustreznosti preskusne metode (21).
If co-elution of such test chemicals occurs with both the cysteine and the lysine peptides then the analysis should be reported as “inconclusive”.
delež vseh negativnih/neaktivnih preskusnih kemikalij, ki se s preskusno metodo pravilno razvrstijo.
In cases where co-elution occurs only with the lysine peptide, then the cysteine 1:10 prediction model reported in Table 2 can be used.
To je merilo točnosti za preskusno metodo, s katero se pridobijo kategorični rezultati, in je pomemben dejavnik pri ocenjevanju ustreznosti preskusne metode.