natgillin/translations-raw huggingface dataset:
However, if there are reasons for using other solvents the scientific rationale should be described.
Matrika odločanja za možne scenarije rezultatov
Cells are exposed to the test chemical by adding 1 μl of the appropriate stock solution in DMSO (see Appendix II to the validation report (4)) per 1 ml supplemented medium (well volume).
Potek preskusa 1
This results in a final concentration of 0,1 % DMSO in the wells.
Potek preskusa 2
To assure adequate mixing it is generally preferred that the appropriate stock solution of the test chemical in DMSO is mixed with supplemented medium to yield the desired final concentration for each dose, and the mixture added to each well immediately after removal of old medium.
Potek preskusa 3
If this option is used, the concentration of DMSO (0,1 %) should remain consistent among all wells.
Odločitev
The wells containing the greatest two concentrations are visually assessed for formation of precipitates or cloudiness as an indication of incomplete solubility of the test chemical by using a stereo microscope.
Scenarij
If such conditions (cloudiness, formation precipitates) are observed, wells containing the next lesser concentrations are examined as well (and so forth) and concentrations that did not completely go into solution are to be excluded from further evaluation and analysis.
Pozitivno
The plate is returned to the incubator at 37 °C under a 5 % CO2 in air atmosphere for 48 hours.
Potrditev [26]
The test chemical plate layout is shown in Table 6.
Konec
Stocks 1 -7 show placement of increasing doses of test chemical.
Izboljšanje [27]
Table 6
Dvoumno [28]
Dosing schematic for the exposure of H295R cells to test chemicals in a 24 well plate
Kontrola kakovosti preskusne plošče
Stock 4
Poleg izpolnjevanja meril kontrole kakovosti plošč je treba izpolniti tudi druga merila kakovosti iz tabele 8, ki se nanašajo na sprejemljive variacije med podvojenimi jamicami, podvojenimi preskusi, linearnostjo in občutljivostjo sistemov za merjenje hormonov, variabilnost med podvojenimi meritvami hormonov istega vzorca in odstotkom izkoristka hormonskih dodatkov po ekstrakciji gojišča (če je primerno; glej odstavek 30 o zahtevah glede ekstrakcije).
Stock 1
Podatki morajo biti razvrščeni v sprejemljiva območja, opredeljena za posamezne parametre, da se upoštevajo pri nadaljnjem vrednotenju.
Stock1
Če ta merila niso izpolnjenja, mora biti v razpredelnici navedeno, da zadevni vzorec ni izpolnjeval meril kontrole kakovosti in da ga je treba ponovno analizirati ali izločiti iz sklopa podatkov.
Stock 5
Tabela 8
Stock 2
Sprejemljiva območja in/ali variacije (%) za parametre preskusne plošče za preskus celic H295R
Stock 6
(LOQ: meja določljivosti sistema za meritev hormonov.
Stock 3
KV: Koeficient variacije; KVT: kontrolni vzorec s topilom; DPM: razgradnje na minuto)
Stock 7
Primerjava med
After 48 hours the exposure plates are removed from the incubator and every well is checked under the microscope for cell condition (attachment, morphology, degree of confluence) and signs of cytotoxicity.
Osnovna proizvodnja hormonov v KVT
The medium from each well is split into two equal amounts (approximately 490 μl each) and transferred to two separate vials appropriately labelled (i.e. one aliquot to provide a spare sample for each well).
Razmerje spremembe, večje od LOQ
To prevent cells from drying out, medium is removed a row or column at a time and replaced with the medium for the cell viability/cytotoxicity assay.
≥ 5-kratno
If cell viability/cytotoxicity is not to be measured immediately, 200 μl PBS with Ca2+ and Mg2+ is added to each well.
≥ 2,5-kratno
The media are frozen at – 80 °C until further processing to analyse hormone concentrations (see paragraphs 44-46).
Preskusi izpostavljenosti – v okviru KV plošče za KVT (podvojene jamice)
While T and E2 in medium kept at – 80 °C are generally stable for at least 3 months, hormone stability during storage should be documented within each laboratory.
Absolutne koncentracije
Immediately after removing the medium, cell viability/cytotoxicity is determined for each exposure plate.
Preskusi izpostavljenosti – med KV plošče za KVT (podvojeni preskusi)
Cell Viability Determination
Faktor spremembe
A cell viability/cytotoxicity assay of choice can be used to determine the potential impact of the test chemical on cell viability.
Sistem merjenja hormonov – občutljivost
The assay should be able to provide a true measure of the percentage of viable cells present in a well, or it should be demonstrated that it is directly comparable to (a linear function of) the Live/Dead® Assay (see Appendix III to the validation report (4)).
Zaznavno razmerje zmanjšanja glede na KVT
An alternative assay that has been shown to work equally well is the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test (18).
Sistem merjenja hormonov – KV podvojene meritve za KVT [29]
The assessment of cell viability using the above methods is a relative measurement that does not necessarily exhibit linear relationships with the absolute number of cells in a well.
Ekstrakcija iz gojišča – izkoristek notranjega standarda 3H (če je primerno)
Therefore, a subjective parallel visual assessment of each well by the analyst should be conducted, and digital pictures of the SCs and the two greatest non-cytotoxic concentrations are to be taken and archived to enable later assessment of true cell density if this should be required.
≥ 65 % nominalno
If by visual inspection or as demonstrated by the viability/cytotoxicity assay there appears to be an increase in cell number, the apparent increase needs to be verified.
ANALIZA PODATKOV IN POROČANJE
If an increase in cell numbers is verified, this should be stated in the test report.
Analiza podatkov
Cell viability will be expressed relative to the average response in the SCs, which is considered 100 % viable cells, and is calculated as appropriate for the cell viability/cytotoxicity assay that is used.
Za vrednotenje relativnega povečanja/zmanjšanja kemijsko spremenjenega proizvajanja hormonov je treba rezultate normalizirati na srednjo vrednost kontrolnega vzorca s topilom vseh preskusnih plošč in rezultate izraziti kot spremembe glede na kontrolne vzorce s topilom na posamezni preskusni plošči.
For the MTT assay, the following formula may be used:
Vse podatke je treba izraziti kot srednjo vrednost ± 1 standardni odklon.
% viable cells = (response in well – average response in MeOH treated [= 100 % dead] wells) ÷ (average response in SC wells – average response in MeOH treated [= 100 % dead] wells)
V analizo podatkov je treba vključiti le podatke o hormonih iz jamic, kjer je bila citotoksičnost manjša od 20 %.
Wells with viability lower than 80 %, relative to the average viability in the SCs (= 100 % viability), should not be included in the final data analysis.
Relativne spremembe je treba izračunati, kot sledi:
Inhibition of steroidogenesis occurring in the presence of almost 20 % cytotoxicity should be carefully evaluated to ensure that cytotoxicity is not the cause for the inhibition.
relativna sprememba = (koncentracija hormonov v posamezni jamici) ÷ (srednja koncentracija hormonov v vseh jamicah s kontrolnim vzorcem s topilom).
Hormone Analysis
Če se na podlagi vizualnega pregleda jamice ali preskusa sposobnosti preživetja/citotoksičnosti iz odstavka 42 zdi, da se je število celic povečalo, je treba to domnevno povečanje preveriti.
Each laboratory can use a hormone measurement system of its choice for the analysis of T and E2.
Pred izvedbo statistične analize je treba opraviti vrednotenje predpostavke normalnosti in homogenosti variance.
Spare aliquots of medium from each treatment group may be used to prepare dilutions to bring the concentration within the linear part of the standard curve.
Normalnost je treba ovrednotiti z uporabo standardnih verjetnostnih grafikonov ali drugih ustreznih statističnih metod (npr. Shapiro-Wilkov preskus).
As noted in paragraph 29, each laboratory should demonstrate the conformance of their hormone measurement system (e.g. ELISA, RIA, LC-MS, LC-MS/MS) with the QC criteria by analysing supplemented medium spiked with an internal hormone control prior to conducting QC runs or testing of chemicals.
Če podatki (razmerja sprememb) niso normalno porazdeljeni, jih je treba poskusiti približati normalni porazdelitvi s pretvorbo.
In order to ensure that the components of the test system do not interfere with measurement of hormones, the hormones may need to be extracted from the media prior to their measurement (see paragraph 30 for the conditions under which an extraction is or is not required).
Če so podatki normalno porazdeljeni ali so blizu normalne porazdelitve, je treba razlike med skupinami koncentracij kemikalije in kontrolnim vzorcem s topilom analizirati z uporabo parametričnega preskusa (npr. Dunnettov preskus), pri čemer je koncentracija neodvisna, odziv (faktor spremembe) pa odvisna spremenljivka.
It is recommended to conduct extraction following the procedures in Appendix III to the validation report (4).
Če so podatki normalno porazdeljeni, je treba uporabiti neparametrični preskus (npr. Kruskal-Wallisov preskus, Steelov preskus).
If a commercial test kit is being used to measure the hormone production, the hormone analysis should be conducted as specified in the manuals provided by the test kit manufacturer.
Razlike se štejejo za značilne, kadar je p ≤ 0,05.
Most manufacturers have a unique procedure by which the hormone analyses are conducted.
Statistična vrednotenja se opravijo na podlagi povprečnih vrednosti posamezne jamice, ki predstavlja neodvisne podatkovne točke s podvojitvami.
Dilutions of samples need to be adjusted such that expected hormone concentrations for the solvent controls fall within the centre of the linear range of the standard curve of the individual assay (Appendix III to the validation report (4)).
Pričakuje se, da zaradi velikega razmika med odmerki v prvem poteku preskusa (skala log10) v veliko primerih ne bo mogoče opisati jasne odvisnosti med koncentracijo in odzivom, kjer bosta dva največja odmerka na linearnem delu sigmoidne krivulje.
Values outside of the linear portion of the standard curve should be rejected.
Zato se za prvi potek preskusa ali kateri koli drug sklop podatkov, pri katerem se pojavi to stanje (npr. kadar ni mogoče oceniti največje učinkovitosti), uporabi statistika s stalno spremenljivko tipa I, opisana zgoraj.
Final hormone concentrations are calculated as follows:
Če na linearnem delu krivulje ležita več kot dve podatkovni točki in kadar je mogoče izračunati največje učinkovitosti, kot je predvideno za nekatere od drugih potekov preskusa, ki se izvedejo z uporabo pollogaritemske razporeditve koncentracij izpostavljenosti, je treba za izračun učinkovitih koncentracij (npr. EC50 in EC20) uporabiti verjetnostni, logaritemski ali drug ustrezen regresijski model.
Example:
Rezultate je treba predstaviti v grafični (palični grafikon, ki predstavlja srednjo vrednost +/– 1 standardni odklon) in tabelarni (najnižja koncentracija z opaženim učinkom/koncentracija brez opaznega učinka, smer učinka in moč največjega odziva, ki je del dela podatkov o odvisnosti med odmerkom in odzivom) obliki (za primer glej sliko 3).
Extracted:
Ocena podatkov se šteje za veljavno le, če je temeljila na vsaj dveh neodvisno izvedenih potekih preskusa.
450 μl medium
Preskus ali potek preskusa se šteje za neodvisen, če je bil izveden na drug dan z uporabo novega kompleta raztopin in kontrolnih vzorcev.
Reconstituted in:
Obseg koncentracij, uporabljen v drugem in tretjem poteku preskusa (če je potrebno), se lahko prilagodi na podlagi rezultatov prvega poteka preskusa, da se bolje opredeli območje odziva na odmerek, ki vsebuje najnižjo koncentracijo z opaženim učinkom (glej odstavek 47).
250 μl assay buffer
Slika 3
Dilution in Assay:
Primer predstavitve in vrednotenja podatkov, pridobljenih med izvajanjem preskusa celic H295R, v grafični in tabelarni obliki
1:10 (to bring the sample within the linear range of the standard curve)
OH Pregnenolon
Hormone Concentration in Assay:
Progesteron
150 pg/ml (already adjusted to concentration per ml sample assayed)
OH Progesteron
Recovery:
Androstenedion
Final hormone concentration =
Testosteron Estron
(Hormone concentration (per ml) ÷ recovery) (dilution factor)
eoksikortikosteron
(150 pg/ml) ÷ (0,89) × (250 μl/450 μl) × 10 = 936,3 pg/ml
Deoksikortizol
Selection of test concentrations
Kortikosteron
A minimum of two independent runs of the assay should be conducted.
Kortizol
Unless prior information such as information on solubility limits or cytotoxicity provides a basis for selecting test concentrations, it is recommended that the test concentrations for the initial run be spaced at log10 intervals with 10–3 M being the maximum concentration.
Aldosteron
If the chemical is soluble, and not cytotoxic at any of the tested concentrations, and the first run was negative for all concentrations, then it is to be confirmed in one more run using the same conditions as the first run was conducted (Table 7).
Kemikalija
If the results of the first run are equivocal (i.e. the fold-change is statistically significant from the SC at only one concentration) or positive (i.e. the fold change at two or more adjacent concentrations is statistically significant), the test should be repeated as indicated in Table 7 by refining the selected test concentrations.
Največja sprememba
Test concentrations in runs two and three (if applicable) should be adjusted on the basis of the results of the initial run bracketing concentrations that elicited an effect using 1/2-log concentration spacing (e.g. if the original run of 0,001, 0,01, 0,1, 1, 10, 100, 1000 μM resulted in inductions at 1 and 10 μM, the concentrations tested in the second run should be 0,1, 0,3, 1, 3, 10, 30, 100 μM), unless lower concentrations need to be employed to achieve a LOEC.
0,15-kratna
In the latter case, at least five concentrations below the lowest concentration tested in the first run should be used in the second run using a 1/2-log scale.
Letrozol
If the second run does not confirm the first run (i.e. statistical significance does not occur at the previously positively tested Live/Deadconcentration ± 1 concentration-increment), a third experiment is to be conducted using the original testing conditions.
ratna
Equivocal results in the first run are considered negative if the observed effect could not be confirmed in any of the two subsequent runs.
Postopek razlage podatkov
Equivocal results are considered as positive responses (effect) when the response can be confirmed in at least one more run within a ± 1 concentration increment (see section 55 for the Data Interpretation Procedure).
Preskusna kemikalija se oceni za pozitivno, če se razmerje indukcije statistično razlikuje (p ≤ 0,05) od kontrolnega vzorca s topilom v dveh sosednjih koncentracijah v vsaj dveh neposredno opravljenih potekih preskusa (tabela 7).
Table 7
Preskusa kemikalija se oceni za negativno na podlagi dveh neodvisnih negativnih potekov preskusa ali v treh potekih preskusa, ki zajemata dva negativna poteka preskusa in en dvoumen ali pozitiven potek preskusa.
Decision matrix for possible outcome scenarios
Če podatki, pridobljeni v treh neodvisnih preskusih, ne izpolnjujejo meril za odločitev iz tabele 7, rezultatov preskusa ni mogoče interpretirati.
Run 1
Rezultati pri koncentracijah, ki presegajo meje topnosti, ali pri citotoksičnih koncentracijah ne smejo biti vključeni v razlago rezultatov.
Run 2
Poročilo o preskusu
Run 3
ime in kraj laboratorija,
Decision
vodjo študije ter drugo osebje in njihove pristojnosti v zvezi s študijo,
Scenario
datum začetka študije in datum konca študije.
Positive
Preskusna kemikalija, reagenti in kontrolni vzorci:
Confirm [26]
identiteto (ime, št.
Stop
CAS, kot je ustrezno), vir, številko lota/serije, čistoto, dobavitelja ter opredelitev preskusne kemikalije, reagentov in kontrolnih vzorcev,
Refine [27]
agregatno stanje in ustrezne fizikalno-kemijske lastnosti preskusne kemikalije,
Equivocal [28]
pogoje shranjevanja ter metodo in pogostost priprave preskusnih kemikalij, reagentov in kontrolnih vzorcev,
Quality Control of the Test Plate
stabilnost preskusne kemikalije.
In addition to meeting the criteria for the QC plate, other quality criteria that pertain to acceptable variation between replicate wells, replicate experiments, linearity and sensitivity of hormone measurement systems, variability between replicate hormone measures of the same sample, and percentage recovery of hormone spikes after extraction of medium (if applicable; see Paragraph 30 regarding extraction requirements) should be met and are provided in Table 8.
Celice:
Data should fall within the acceptable ranges defined for each parameter to be considered for further evaluation.
vir in vrsto celic,
Table 8
opis postopkov za ohranitev celičnih kultur.
Acceptable ranges and/or variation (%) for H295R assay test plate parameters.
Zahteve pred preskušanjem (če je ustrezno):
(LOQ: Limit of Quantification of the hormone measurement system.
opis in rezultate preskusa vpliva kemikalij na preskus hormonov,
CV: Coefficient of variation; SC: Solvent Control; DPM: Disintegrations per minute)
opis in rezultate meritev učinkovitosti ekstrakcije hormonov,
Comparison Between
umeritvene in umeritvene krivulje za vse analitične preskuse, ki jih je treba izvesti,
Basal hormone production in SCs
meje zaznavnosti za izbrane analitične preskuse.
Fold-greater than LOQ
Preskusni pogoji:
≥ 5-fold
sestavo gojišča,
≥ 2,5-fold
koncentracijo preskusne kemikalije,
Exposure Experiments — Within Plate CV for SCs (Replicate Wells)
gostoto celic (ocenjena ali izmerjena koncentracija celic po 24 urah in 48 urah),
Absolute Concentrations
topnost preskusne kemikalije (meja topnosti, če je določena),
Exposure Experiments — Between Plate CV for SCs (Replicate Experiments)
čas in pogoje inkubacije.
Fold-Change
Rezultati preskusa:
Hormone Measurement System — Sensitivity
neobdelane podatke za vse jamice za kontrolne vzorce in preskusne kemikalije – vsako podvojeno meritev v obliki izvirnih podatkov, pridobljenih z instrumentom, uporabljenim za meritev proizvajanja hormonov (npr. optična gostota, enote fluorescence, razgradnja na minuto itd.),
Detectable fold-decrease relative to SC
validacijo normalnosti ali razlago pretvorbe podatkov,
Hormone Measurement System — Replicate Measure CV for SCs [29]
srednjo vrednost odzivov ± 1 standardni odklon za vsako izmerjeno jamico,
Medium Extraction — Recovery of Internal 3H Standard (If Applicable)
podatke o citotoksičnosti (preskusne koncentracije, ki so povzročile citotoksičnost),
DPM
potrditev, da so bile zahteve glede kontrole kakovosti izpolnjene,
≥ 65 % Nominal
relativno spremembo v primerjavi s kontrolnim vzorcem s topilom, s popravkom za citotoksičnost,
DATA ANALYSIS AND REPORTING
palični grafikon, ki kaže relativno spremembo (razmerje spremembe) pri posamezni koncentraciji, standardni odklon in statistično značilnost, kot je navedeno v odstavkih 49–54.
Data Analysis
Razlaga podatkov:
To evaluate the relative increase/decrease in chemically altered hormone production, the results should be normalised to the mean SC value of each test plate, and results expressed as changes relative to the SC in each test plate.
uporabo postopka za razlago podatkov za rezultate in razpravo o ugotovitvah.
All data are to be expressed as mean ± 1 standard deviation (SD).
Razprava:
Only hormone data from wells where cytotoxicity was less than 20 % should be included in the data analysis.
ali v študiji obstajajo znaki za možnost, da bi lahko na podatke o T/E2 vplivali posredni učinki na glukokortikoidne in mineralokortikoidne poti?
Relative changes should be calculated as follows:
Sklepne ugotovitve
If data are not normally distributed, an appropriate non-parametric test should be used (e.g. Kruskal Wallis, Steel's Many-one rank test).
Poglavje B.55 te priloge:
Differences are considered significant at p ≤ 0,05.
Hershbergerjev biološki preskus na podganah:
Statistical evaluations are done based on average values for each well that represent independent replicate data points.
kratkotrajni presejalni preskus za (anti)androgene lastnosti.
It is anticipated that due to the large spacing of doses in the first run (log10 scale) in many cases it will not be possible to describe clear concentration-response relationships where the two greatest doses will be on the linear portion of the sigmoid curve.
Dodatek
Therefore, for the first run or any other data sets where this condition occurs (e.g. where no maximum efficacy can be estimated) type I fixed variable statistics as described above will be applied.
Konfluentnost se nanaša na pokritje ali množitev, ki je celicam dovoljena v gojišču kulture ali čez njega.
Results should be provided both in graphical (bar graphs representing mean ± 1 SD) and tabular (LOEC/NOEC, direction of effect, and strength of maximum response that is part of the dose-response portion of the data) formats (see Figure 3 for an example).
CYP pomeni citokrom P450 mono-oksigenaze, družino genov in encimov, ki jih proizvajajo, ki sodelujejo pri katalizi različnih biokemijskih reakcij, vključno s sintezo in presnovo steroidnih hormonov.
Data assessment is only considered valid if it has been based on at least two independently conducted runs.
DPM pomeni število razgradenj na minuto.
An experiment or run is considered independent if it has been conducted at a different date using a new set of solutions and controls.
To je število atomov v dani količini radioaktivne snovi, za katero se zazna, da razpade v eni minuti.
The concentration range used in runs 2 and 3 (if necessary) may be tailored on the basis of the results of run 1 to better define the dose response range containing the LOEC (see paragraph 47).
E2 je 17β-estradiol, tj. najpomembnejši estrogen v sistemih sesalcev.
Figure 3
Celice H295R so človeške adenokarcinomske celice, ki imajo fiziološke lastnosti consko nediferenciranih človeških fetalnih celic nadledvičnice in izražajo vse encime steroidogenezne poti.
Example of the presentation and evaluation of data obtained during the conduct of the H295R Assay in graphical and tabular format.
Zagotavlja jih ATCC.
Asterisks indicate statistically significant differences from the solvent control (p < 0,05).
Gojišče za zamrzovanje se uporablja za zamrzovanje in shranjevanje zamrznjenih celic.
Lowest observed effective concentration;
Zajema založno gojišče ter BD Nu-Serum in dimetilsulfoksid.
Max Change:
Linearni razpon je razpon v okviru umeritvene krivulje za sistem meritve hormonov, kjer so rezultati sorazmerni s koncentracijo analita, prisotnega v vzorcu.
Maximum strength of the response observed at any concentration relative to the average SC response (= 1).
LOQ je kratica za 'mejo določljivosti' in je najmanjša količina kemikalije, ki jo je mogoče razločiti od odsotnosti kemikalije (slepa vrednost) v dani meji zaupanja.
Cholesterol
Za namen te metode mejo določljivosti običajno opredeli proizvajalec preskusnih sistemov, če ni določeno drugače.
Pregnenolone
LOEC je najnižja koncentracija z opaženim učinkom, tj. najmanjša raven koncentracije, pri kateri se odziv pri preskusu statistično razlikuje od odziva kontrolnega vzorca s topilom.
OH Pregnenolone
NOEC je koncentracija brez opaznega učinka, ki je najvišja preskušena koncentracija, če pri preskusu ni pozitivnega odziva.
Progesterone
Pasaža je število delitev celic po iniciaciji kulture iz zamrznjene zaloge.
OH Progesterone
Začetna pasaža, ki se je začela iz zamrznjene zaloge, ima številko ena (1).
Androstenedione
Celice, ki so se delile enkrat, so označene kot pasaža 2 itd.
Testosterone
PBS je Dulbeccov fosfatni pufer s soljo.
estradiol
Kontrola kakovosti, okrajšano QC, pomeni ukrepe, ki so potrebni za zagotovitev veljavnih podatkov.
eoxycorticosterone
Plošča za kontrolo kakovosti je plošča s 24 jamicami, ki vsebuje dve koncentraciji pozitivnih in negativnih kontrolnih vzorcev za spremljanje uspešnosti nove serije celic ali zagotavljanje pozitivnih kontrolnih vzorcev za preskušanje kemikalij.
Cortisol
Ponovitev je neodvisen preskus z novim kompletom raztopin in kontrolnih vzorcev.
Aldosterone
Založno gojišče je osnova za pripravo drugih reagentov.