natgillin/translations-raw huggingface dataset:
However, long term treatment (without S9) should have its own positive control, as the treatment duration will differ from the test using metabolic activation.
Nato se šteje, da preskusna kemikalija ne more povzročiti preloma kromosomov in/ali pridobitve ali izgube v tem preskusnem sistemu.
If a clastogen is selected as the single positive control for short-term treatment with and without metabolic activation, an aneugen should be selected for the long-term treatment without metabolic activation.
Preverjanje jasno pozitivnega in jasno negativnega odziva ni potrebno.
Positive controls for both clastogenicity and aneugenicity should be used in metabolically competent cells that do not require S9.
Če odziv ni niti jasno negativen niti jasno pozitiven, kot je opisano zgoraj, in/ali za podporo pri ugotavljanju biološke pomembnosti rezultatov, je treba podatke oceniti s strokovno presojo in/ali nadaljnjimi preiskavami.
Each positive control should be used at one or more concentrations expected to give reproducible and detectable increases over background in order to demonstrate the sensitivity of the test system (i.e. the effects are clear but do not immediately reveal the identity of the coded slides to the reader), and the response should not be compromised by cytotoxicity exceeding the limits specified in this test method.
Štetje dodatnih celic (po potrebi) ali izvedba ponovitve poskusa je lahko koristna, po možnosti z uporabo spremenjenih preskusnih pogojev (npr. razmik med koncentracijami, drugi pogoji za presnovno aktivacijo [tj. koncentracija ali izvor S9]).
Reference chemicals recommended for assessing laboratory proficiency and for the selection of positive controls
Zbirka podatkov v redkih primerih celo po izvedbi nadaljnjih preiskav onemogoča sprejetje sklepa o pozitivnih ali negativnih rezultatih, zato se zaključi, da je odziv dvoumen.
Aneugens
Preskusne kemikalije lahko pri in vitro preskusu mikronukleusov povzročijo nastanek mikronukleusov zato, ker povzročajo prelom ali izgubo kromosomov ali pa oboje hkrati.
Colchicine
Za ugotovitev, ali je mehanizem za nastajanje mikronukleusov posledica klastogenega in/ali anevgenega delovanja, se lahko izvede nadaljnja analiza z uporabo antikinetohornih protiteles, in situ sond, specifičnih za centromere, ali drugih metod.
Vinblastine
reaktivnost preskusne kemikalije s topilom/vehiklom ali gojiščem celic;
Treatment Schedule
topnost in stabilnost preskusne kemikalije v topilu/nosilcu, če sta znani;
In order to maximise the probability of detecting an aneugen or clastogen acting at a specific stage in the cell cycle, it is important that sufficient numbers of cells representing all of the various stages of their cell cycles are treated with the test chemical.
meritve vrednosti pH, osmolarnosti in oborine v gojišču, ki mu je bila dodana preskusna kemikalija, če je ustrezno.
All treatments should commence and end while the cells are growing exponentially and the cells should continue to grow up to the time of sampling.
utemeljitev izbire topila;
The treatment schedule for cell lines and primary cell cultures may, therefore, differ somewhat from that for lymphocytes which require mitogenic stimulation to begin their cell cycle (17).
odstotek topila v končnem gojišču.
For lymphocytes, the most efficient approach is to start the treatment with the test chemical at 44-48 hours after PHA stimulation, when cells will be dividing asynchronously (6).
vrsta in izvor uporabljenih celic;
Published data (19) indicate that most aneugens and clastogens will be detected by a short term treatment period of 3 to 6 hours in the presence and absence of S9, followed by removal of the test chemical and sampling at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (7).
ustreznost uporabljene vrste celic;
However, for thorough evaluation, which would be needed to conclude a negative outcome, all three following experimental conditions should be conducted using a short term treatment with and without metabolic activation and long term treatment without metabolic activation (see paragraphs 56, 57 and 58): Cells should be exposed to the test chemical without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (19),
za celične linije – odsotnost mikoplazme;
Cells should be exposed to the test chemical with metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (19),
za celične linije – informacije o trajanju celičnega cikla, podvojitvenem času ali indeksu proliferacije;
Cells should be continuously exposed without metabolic activation until sampling at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths.
pri uporabi limfocitov – spol donorjev krvi, starost in vse pomembne informacije o donorju, uporabljeni krvi v celoti ali ločenih limfocitih, mitogenih;
If it is known or suspected that the test chemical affects the cell cycling time (e.g. when testing nucleoside analogues), especially for p53 competent cells (35) (36) (77), sampling or recovery times may be extended by up to a further 1,5 - 2,0 normal cell cycle lengths (i.e. total 3,0 to 4,0 cell cycle lengths after the beginning of short-term and long-term treatments).
običajno trajanje celičnega cikla (negativne kontrole);
These options address situations where there may be concern regarding possible interactions between the test chemical and cytoB.
za celične linije – metode vzdrževanja celičnih kultur;
When using extended sampling times (i.e. total 3,0 to 4,0 cell cycle lengths culture time), care should be taken to ensure that the cells are still actively dividing.
za celične linije – modalno število kromosomov.
For example, for lymphocytes exponential growth may be declining at 96 hours following stimulation and monolayer cultures of cells may become confluent.
Preskusni pogoji:
The suggested cell treatment schedules are summarised in Table 2.
identiteta snovi za citogenetski blok (npr. cytoB), če je uporabljena, njena koncentracija in trajanje izpostavljenosti celic;
These general treatment schedules may be modified (and should be justified) depending on the stability or reactivity of the test chemical or the particular growth characteristics of the cells being used.
koncentracija preskusne kemikalije, izražena kot končna koncentracija v gojišču (npr. v μg ali mg/ml ali mM gojišča);
Cell treatment and harvest times for the MNvit test
razlogi za izbiro koncentracij in števila kultur, vključno s podatki o citotoksičnosti in mejah topnosti;
Lymphocytes, primary cells and cell lines treated with cytoB
sestava gojišča, koncentracija CO2, če je na voljo, stopnja vlažnosti;
Treat for 3-6 hours in the presence of S9;
inkubacijska temperatura in čas trajanja inkubacije;
remove the S9 and treatment medium;
gostota celic ob nasaditvi, če je ustrezno;
add fresh medium and cytoB;
vrsta in sestava sistema presnovne aktivacije (vir S9, način priprave zmesi S9, koncentracija ali volumen zmesi S9, S9 v končnem gojišču, nadzor kakovosti S9 (npr. encimska aktivnost, sterilnost, presnovna zmogljivost);
harvest 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment.
kemikalije pozitivne in negativne kontrole, končne koncentracije, pogoji in trajanje tretiranja ter obdobja obnavljanja;
Treat for 3-6 hours;
metode priprave preparatov in uporabljene tehnike barvanja;
harvest at the end of the treatment period.
uporabljene metode statistične analize;
Cell lines treated without cytoB
metode, kot so uporaba antikinetohornih protiteles ali pancentromerno specifičnih sond, da se ugotovi, ali mikronukleusi vsebujejo cele kromosome ali njihove fragmente, če je ustrezno;
(Identical to the treatment schedules outlined above with the exception that no cytoB is added)
opredelitev celic, sprejemljivih za analizo;
For monolayer cultures, mitotic cells (identifiable as being round and detaching from the surface) may be present at the end of the 3-6 hour treatment.
ob odsotnosti citohalazina B število tretiranih celic in število odvzetih celic za vsako kulturo v primeru celičnih linij;
If there is evidence for a substantial increase in the number of mitotic cells compared with controls, indicating likely mitotic arrest, then the cells should be collected by centrifugation and added back to the culture, to avoid losing cells that are in mitosis, and at risk for micronuclei/chromosome aberration, at the time of harvest.
uporabljena meritev citotoksičnosti, npr. proliferacijski indeks citokinetskega bloka ali indeks pomnoževanja v primeru metode citokinetskega bloka; relativno povečanje števila celic ali relativna podvojitev populacije, če se metode citokinetskega bloka ne uporabljajo; morebitna druga opažanja (npr. konfluentnost celic, apoptoza, nekroza, štetje v metafazi, pogostost celic z dvema jedroma);
Cell harvest and slide preparation
podatki o vrednosti pH in osmolarnosti obdelovalnega gojišča, če sta bili določeni;
Each culture should be harvested and processed separately.
porazdelitev celic z enim, dvema ali več jedri v primeru uporabe metode citokinetskega bloka;
Cell preparation may involve hypotonic treatment, but this step is not necessary if adequate cell spreading is otherwise achieved.
število celic z mikronukleusi, navedeno ločeno za vsako tretirano in kontrolno kulturo, ter po potrebi opredelitev, ali so iz celic z dvema ali enim jedrom;
Different techniques can be used in slide preparation provided that high-quality cell preparations for scoring are obtained.
podatki o negativnih (s topilom) in pozitivnih kontrolah iz preteklih preskusov, skupaj z razponi, srednjimi vrednostmi in standardnimi odkloni ter 95-odstotnimi kontrolnimi mejami za porazdelitev, pa tudi število podatkov;
Cells with intact cell membrane and intact cytoplasm should be retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells.
statistična analiza; p-vrednosti, če obstajajo.
The slides can be stained using various methods, such as Giemsa or fluorescent DNA specific dyes.
Poglavje B.10 te priloge: In vitro preskus kromosomskih aberacij pri sesalcih.
The use of appropriate fluorescent stains (e.g. acridine orange (78) or Hoechst 33258 plus pyronin-Y (79)) can eliminate some of the artifacts associated with using a non-DNA specific stain.
In vitro micronucleus assay with Chinese hamster V79 cells – results of a collaborative study with in situ exposure to 26 chemical substances.
Anti-kinetochore antibodies, FISH with pancentromeric DNA probes, or primed in situ labelling with pancentromere-specific primers, together with appropriate DNA counterstaining, can be used to identify the contents (whole chromosomes will be stained while acentric chromosome fragments will not) of micronuclei if mechanistic information of their formation is of interest (16) (17).
Mutation Research, zv.
Other methods for differentiation between clastogens and aneugens may be used if they have been shown to be effective and validated.
A protocol for the in vitro micronucleus test, I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity.
For example, for certain cell lines the measurements of sub-2N nuclei as hypodiploid events using techniques such as image analysis, laser scanning cytometry or flow cytometry could also provide useful information (80) (81) (82).
Mutation Research, zv.
Morphological observations of nuclei could also give indications of possible aneuploidy.
Evaluation of different cytotoxic and cytostatic measures for the in vitromicronucleus test (MNVit): Introduction to the collaborative trial.
Moreover, a test for metaphase chromosome aberrations, preferably in the same cell type and protocol with comparable sensitivity, could also be a useful way to determine whether micronuclei are due to chromosome breakage (knowing that chromosome loss would not be detected in the chromosome aberration test).
Mutation Research, zv.
All slides, including those of the solvent and the untreated (if used) and positive controls, should be independently coded before the microscopic analysis of micronucleus frequencies.
Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells.
Appropriate techniques should be used to control any bias or drift when using an automated scoring system, for instance, flow cytometry, laser scanning cytometry or image analysis.
Regulatory Toxicology and Pharmacology, zv.
Regardless of the automated platform is used to enumerate micronuclei, CBPI, RI, RPD, or RICC should be assessed concurrently.
Statement by the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) on the scientific validity of the in vitro micronucleus test as an alternative to the in vitro chromosome aberration assay for genotoxicity testing.
In cytoB-treated cultures, micronucleus frequencies should be analysed in at least 2000 binucleate cells per concentration and control (83), equally divided among the replicates, if replicates are used.
sestanek odbora ESAC, 16.–17. novembra 2006.
In the case of single cultures per dose (see paragraph 28), at least 2000 binucleate cells per culture (83) should be scored in this single culture.
Na voljo na: http://ecvam.jrc.it/index.htm.
If substantially fewer than 1000 binucleate cells per culture (for duplicate cultures), or 2000 (for single culture), are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test should be repeated using more cells, or at less cytotoxic concentrations, whichever is appropriate.
Na voljo na: http://ecvam.jrc.it/index.htm.
Care should be taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size.
„A safe substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems“, v In Vitro Metabolic Activation in Mutagenesis Testing. de Serres, F. J. idr.
In addition, binucleate cells should not be confused with poorly spread multi-nucleate cells.
(eds.), Elsevier, North-Holland, str 85–88.
Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells (84).
Na voljo na: http://www.pops.int/.
Scoring of mononucleate cells is acceptable if the test chemical is shown to interfere with cytoB activity.
Corrigendum to „Report from the in vitro micronucleus assay working group“.
A repeat test without CytoB might be useful in such cases.
Mutation Research, zv.
Scoring mononucleate cells in addition to binucleate cells could provide useful information (85) (86), but is not mandatory.
OECD Environment, Health and Safety Publications (EHS), Series on testing and assessment, št. 198, OECD Publishing, Pariz.
In cell lines tested without cytoB treatment, micronuclei should be scored in at least 2000 cells per test concentration and control (83), equally divided among the replicates, if replicates are used.
„In vitro micronucleus test“, in Encyclopedia of Biopharmaceutical Statistics, druga izdaja, Chow, S. (ed.), Marcel Dekker, Inc.
When single cultures per concentration are used (see paragraph 28), at least 2000 cells per culture should be scored in this single culture.
New York, str. 463–467.
If substantially fewer than 1000 cells per culture (for duplicate cultures), or 2000 (for single culture), are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test should be repeated using more cells, or at less cytotoxic concentrations, whichever is appropriate.
Anevgen
When cytoB is used, a CBPI or an RI should be determined to assess cell proliferation (see Appendix 2) using at least 500 cells per culture.
vsaka kemikalija ali proces, na podlagi katerega pride do anevploidije v celicah ali organizmih zaradi reagiranja z elementi mitotskega in mejoznega cikla delitve celic.
When treatments are performed in the absence of cytoB, it is essential to provide evidence that the cells in culture have divided, as discussed in paragraphs 24-28.
programirana celična smrt, za katero je značilna vrsta korakov, ki vodijo v razgradnjo celic na delce, obdane z membrano, ki se nato odstranijo s fagocitozo ali osipanjem.
In order to establish sufficient experience with the assay prior to using it for routine testing, the laboratory should have performed a series of experiments with reference positive chemicals acting via different mechanisms (at least one with and one without metabolic activation, and one acting via an aneugenic mechanism, and selected from the chemicals listed in Table 1) and various negative controls (including untreated cultures and various solvents/vehicle).
povečanje števila celic kot posledica mitotične delitve celic.
This is not applicable to laboratories that have experience, i.e. that have an historical data base available as defined in paragraphs 49 to 52.
območje kromosoma, ki vsebuje DNK, kjer sta spojeni obe kromatidi in na katerem sta na obeh straneh pritrjeni obe kinetohori.
A selection of positive control chemicals (see Table 1) should be investigated with short and long treatments in the absence of metabolic activation, and also with short treatment in the presence of metabolic activation, in order to demonstrate proficiency to detect clastogenic and aneugenic chemicals, determine the effectiveness of the metabolic activation system and demonstrate the appropriateness of the scoring procedures (microscopic visual analysis, flow cytometry, laser scanning cytometry or image analysis).
se nanašajo na končne koncentracije preskusne kemikalije v gojišču.
When first acquiring data for an historical negative control distribution, concurrent negative controls should be consistent with published negative control data where they exist.
vsaka kemikalija, ki povzroči strukturne kromosomske aberacije v populacijah celic ali evkariotskih organizmih.
As more experimental data are added to the control distribution, concurrent negative controls should ideally be within the 95 % control limits of that distribution (87) (88).
Citokineza
The laboratory's historical negative control database, should initially be built with a minimum of 10 experiments but would preferably consist of at least 20 experiments conducted under comparable experimental conditions.
proces delitve celic, ki nastopi takoj po mitozi in vpliva na oblikovanje dveh hčerinskih celic, ki imata vsaka eno samo jedro.
Laboratories should use quality control methods, such as control charts (e.g. C-charts or X-bar charts (88)), to identify how variable their positive and negative control data are, and to show that the methodology is ‘under control’ in their laboratory (83).
Proliferacijski indeks citokinetskega bloka (Cytokinesis-Block Proliferation index – CPBI)
Further recommendations on how to build and use the historical data (i.e. criteria for inclusion and exclusion of data in historical data and the acceptability criteria for a given experiment) can be found in the literature (87).
delež celic, pri katerih se je izvedla druga delitev, v tretirani populaciji glede na netretirano kontrolo (glej Dodatek 2 za formulo).
Any changes to the experimental protocol should be considered in terms of the consistency of the data with the laboratory's existing historical control databases.
Citostaza
Negative control data should consist of the incidence of micronucleated cells from a single culture or the sum of replicate cultures as described in paragraph 28.
zaviranje rasti celic (glej Dodatek 2 za formulo).
Concurrent negative controls should ideally be within the 95 % control limits of the distribution of the laboratory's historical negative control database (87) (88).
Za poskuse, zajete v to preskusno metodo in izvedene v prisotnosti citohalazina B, je citotoksičnost opredeljena kot zmanjšanje proliferacijskega indeksa citokinetskega bloka (CBPI) ali indeksa pomnoževanja pri tretiranih celicah v primerjavi z negativno kontrolo (glej odstavek 26 in Dodatek 2).
Where concurrent negative control data fall outside the 95 % control limits, they may be acceptable for inclusion in the historical control distribution as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ (see paragraph 50) and there is evidence of absence of technical or human failure.
Za poskuse, zajete v to preskusno metodo in izvedene brez citohalazina B, je citotoksičnost opredeljena kot zmanjšanje relativne podvojitve populacije (RPD) ali relativnega povečanja števila celic (RICC) pri tretiranih celicah v primerjavi z negativno kontrolo (glej odstavek 27 in Dodatek 2).
If the cytokinesis-block technique is used, only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) are used in the evaluation of micronucleus induction.
Celice v interfazi
The scoring of the numbers of cells with one, two, or more micronuclei can be reported separately and could provide useful information, but is not mandatory.
celice, ki niso v fazi mitoze.
Concurrent measures of cytotoxicity and/or cytostasis for all treated, negative and positive control cultures should be determined (16).
beljakovinska struktura, ki nastane na centromeri kromosoma, na katero se med delitvijo celice pripnejo niti delitvenega vretena, kar omogoča pravilno gibanje hčerinskih kromosomov proti poloma hčerinskih celic.
The CBPI or the RI should be calculated for all treated and control cultures as measurements of cell cycle delay when the cytokinesis-block method is used.
majhna jedra, poleg glavnega jedra celic in ločena od njega, ki nastanejo med telofazo mitoze ali mejoze iz zaostalih fragmentov kromosomov ali celih kromosomov.
In the absence of cytoB, the RPD or the RICC should be used (see Appendix 2).
razmerje med številom celic v metafazi in skupnim številom celic v opazovani populaciji celic; kazalnik stopnje celične proliferacije te populacije.
The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database as described in paragraph 50.
Nerazdvajanje
Concurrent positive controls (see paragraph 50) should induce responses that are compatible with those generated in the laboratory's historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
par kromatid se ne razdvoji in ustrezno loči v razvijajoče se hčerinske celice, zaradi česar imata hčerinski celici nenormalno število kromosomov.
Cell proliferation criteria in the solvent control should be fulfilled (paragraph 25-27).
numerične kromosomske aberacije v celicah ali organizmih, ki ne vplivajo na posamezen kromosom ali kromosome (anevploidija), temveč na celoten set kromosomov.
All experimental conditions were tested unless one resulted in positive results (paragraphs 36-40).
Proliferacijski indeks
Adequate number of cells and concentrations are analysable (paragraphs 28 and 44-46).
metoda merjenja citotoksičnosti, ko se citohalazin B ne doda (glej Dodatek 2 za formulo).
The criteria for the selection of top concentration are consistent with those described in paragraphs 24-31.
Relativno povečanje števila celic (RICC)
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined (see paragraphs 36-39):
Indeks pomnoževanja (RI)
at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (89)
razmerje ciklov celične delitve v tretirani kulturi glede na netretirano kulturo med obdobjem izpostavljenosti in obnavljanja (glej Dodatek 2 za formulo).
the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test (see paragraph 28)
mešanica S9, pripravljene iz jeter, in kofaktorjev, potrebnih za dejavnosti presnovnih encimov.