natgillin/translations-raw huggingface dataset:
This test method is equivalent to OECD test guideline 474 (2016).
Pri obdobju odmerjanja, krajšem od 14 dni, je mejni odmerek 2000 mg/kg telesne teže na dan.
The mammalian in vivo micronucleus test is especially relevant for assessing genotoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA repair processes are active and contribute to the responses.
Zato se lahko ob ustrezni utemeljitvi izberejo načini izpostavljenosti, kot so s prehrano, pitno vodo, topično podkožno, intravenozno, oralno (z gavažo), z vdihavanjem, intratrahealno ali z vsadkom.
The mammalian in vivo micronucleus test is used for the detection of damage induced by the test chemical to the chromosomes or the mitotic apparatus of erythroblasts.
Če je preskusna kemikalija primešana hrani ali pitni vodi, zlasti v primeru enega samega odmerka, je treba paziti, da med zaužitjem hrane in vode ter vzorčenjem preteče dovolj časa, da se lahko odkrijejo učinki (glej odstavek 37).
The test evaluates micronucleus formation in erythrocytes sampled either in the bone marrow or peripheral blood cells of animals, usually rodents.
Po možnosti se izvedeta 2 tretiranji ali več s preskusno kemikalijo, ki se daje v 24-urnih intervalih, zlasti kadar je ta preskus vključen v druge študije toksičnosti.
The purpose of the micronucleus test is to identify chemicals that cause cytogenetic damage which results in the formation of micronuclei containing either lagging chromosome fragments or whole chromosomes.
Druga možnost je dajanje posamičnih odmerkov, če je to znanstveno utemeljeno (npr. če je za preskusne kemikalije znano, da zavirajo celični cikel).
When a bone marrow erythroblast develops into an immature erythrocyte (sometimes also referred to as a polychromatic erythrocyte or reticulocyte), the main nucleus is extruded; any micronucleus that has been formed may remain behind in the cytoplasm.
Preskusne kemikalije se lahko dajejo tudi v razdeljenem odmerku, kar pomeni dva ali več odmerkov v istem dnevu na največ 2 do 3 ure, s čimer se olajša dajanje velike količine kemikalije.
Visualisation or detection of micronuclei is facilitated in these cells because they lack a main nucleus.
Preskus na miših ali podganah se lahko izvede na enega od naslednjih treh načinov:
An increase in the frequency of micronucleated immature erythrocytes in treated animals is an indication of induced structural or numerical chromosomal aberrations.
a.
Newly formed micronucleated erythrocytes are identified and quantitated by staining followed by either visual scoring using a microscope, or by automated analysis.
Živali se s preskusno kemikalijo tretirajo enkrat.
Counting sufficient immature erythrocytes in the peripheral blood or bone marrow of adult animals is greatly facilitated by using an automated scoring platform.
Vzorci kostnega mozga se odvzamejo najmanj dvakrat (iz neodvisnih skupin živali), in sicer vsaj 24 ur in najpozneje 48 ur po tretiranju, z ustreznimi intervali med vzorci, razen če je znano, da ima kemikalija izjemno dolg razpolovni čas.
Such platforms are acceptable alternatives to manual evaluation (2).
Vzorčenje, izvedeno prej kot 24 ur po tretiranju, je treba utemeljiti.
Comparative studies have shown that such methods, using appropriate calibration standards, can provide better inter- and intra-laboratory reproducibility and sensitivity than manual microscopic scoring (3) (4).
Vzorci periferne krvi se odvzamejo vsaj dvakrat (iz iste skupine živali), in sicer vsaj 36 ur in najpozneje 72 ur po tretiranju, z ustreznimi intervali med vzorčenji.
Automated systems that can measure micronucleated erythrocyte frequencies include, but are not limited to, flow cytometers (5), image analysis platforms (6) (7), and laser scanning cytometers (8).
Če se pri enem času vzorčenja odkrije pozitiven odziv, dodatno vzorčenje ni potrebno, razen če so potrebne kvantitativne informacije o razmerju med odmerkom in odzivom.
Although not normally done as part of the test, chromosome fragments can be distinguished from whole chromosomes by a number of criteria.
Opisani odvzem celic je posledica kinetike pojava in izginjanja mikronukleusov v teh dveh tkivnih prostorih.
These include identification of the presence or absence of a kinetochore or centromeric DNA, both of which are characteristic of intact chromosomes.
b.
The absence of kinetochore or centromeric DNA indicates that the micronucleus contains only fragments of chromosomes, while the presence is indicative of chromosome loss.
Pri dveh dnevnih odmerkih (kar pomeni dva odmerka na 24 ur) je treba vzorce kostnega mozga odvzeti enkrat med 18 in 24 ur po zadnjem tretiranju, vzorce periferne krvi pa enkrat med 36 in 48 ur po zadnjem tretiranju (30).
The bone marrow of young adult rodents is the target tissue for genetic damage in this test since erythrocytes are produced in this tissue.
c.
The measurement of micronuclei in immature erythrocytes in peripheral blood is acceptable in other mammalian species for which adequate sensitivity to detect chemicals that cause structural or numerical chromosomal aberrations in these cells has been demonstrated (by induction of micronuclei in immature erythrocytes) and scientific justification is provided.
Pri treh ali več dnevnih odmerkih (kar pomeni tri ali več odmerkov na približno 24 ur) je treba vzorce kostnega mozga odvzeti najpozneje 24 ur po zadnjem tretiranju, vzorce periferne krvi pa najpozneje 40 ur po zadnjem tretiranju (31).
The frequency of micronucleated immature erythrocytes is the principal endpoint.
V to možnost tretiranja je vključena kombinacija kometnega preskusa (kar pomeni vzorčenje 2 do 6 ur po zadnjem tretiranju) s preskusom mikronukleusov in vključitev preskusa mikronukleusov v študijo toksičnosti s ponavljajočimi se odmerki.
The frequency of mature erythrocytes that contain micronuclei in the peripheral blood also can be used as an endpoint in species without strong splenic selection against micronucleated cells and when animals are treated continuously for a period that exceeds the lifespan of the erythrocyte in the species used (e.g. 4 weeks or more in the mouse).
Iz zbranih podatkov je bilo razvidno, da je v teh širših časovnih okvirih pri 3 ali več odmerkih kemikalije opaziti nastajanje mikronukleusov (15).
Animals are exposed to the test chemical by an appropriate route.
Uporabljajo se lahko tudi drugi režimi odmerjanja in vzorčenja, če so ustrezni in znanstveno utemeljeni, da se olajša povezovanje z drugimi študijami toksičnosti.
If bone marrow is used, the animals are humanely euthanised at an appropriate time(s) after treatment, the bone marrow is extracted, and preparations are made and stained (9) (10) (11) (12) (13) (14) (15).
Vse živali je treba stehtati ob začetku študije, vsaj enkrat na teden med študijami s ponavljajočimi se odmerki in ob evtanaziji.
When peripheral blood is used, the blood is collected at an appropriate time(s) after treatment and preparations are made and stained (12) (16) (17) (18).
Živali, ki kažejo neletalne znake čezmerne toksičnosti, je treba humano evtanazirati pred koncem preskusnega obdobja (28).
When treatment is administered acutely, it is important to select bone marrow or blood harvest times at which the treatment-related induction of micronucleated immature erythrocytes can be detected.
Pod določenimi pogoji se lahko spremlja telesna temperatura živali, saj sta v nastajanje napačnih rezultatov vključeni hiper- in hipotermija, ki ju povzroči tretiranje (32) (33) (34).
In the case of peripheral blood sampling, enough time must also have elapsed for these events to appear in circulating blood.
Ob ustreznih časih je treba odvzeti vzorec krvi, da se lahko preišče raven preskusnih kemikalij v plazmi, s čimer se dokaže, da je prišlo do izpostavljenosti kostnega mozga, če je to utemeljeno in če ne obstajajo drugi podatki o izpostavljenosti (glej odstavek 48).
Preparations are analysed for the presence of micronuclei, either by visualisation using a microscope, image analysis, flow cytometry, or laser scanning cytometry.
Priprava kostnega mozga/krvi
In order to establish sufficient experience with the conduct of the assay prior to using it for routine testing, the laboratory should have demonstrated the ability to reproduce expected results from published data (17) (19) (20) (21) (22) for micronucleus frequencies with a minimum of two positive control chemicals (including weak responses induced by low doses of positive controls), such as those listed in Table 1 and with compatible vehicle/solvent controls (see paragraph 26).
Takoj po humani evtanaziji se celice kostnega mozga običajno pridobijo iz stegnenic ali golenic živali.
These experiments should use doses that give reproducible and dose-related increases and demonstrate the sensitivity and dynamic range of the test system in the tissue of interest (bone marrow or peripheral blood) and using the scoring method to be employed within the laboratory.
Celice se običajno odstranijo, preparirajo in obarvajo z uporabo uveljavljenih metod.
This requirement is not applicable to laboratories that have experience, i.e. that have a historical database available as defined in paragraphs 14-18.
Majhne količine periferne krvi se lahko v skladu z ustreznimi standardi dobrobiti živali pridobijo z metodo, ki omogoča preživetje poskusne živali, kot je s puščanjem krvi iz repne vene ali druge primerne žile, ali pa s punkcijo srca ali vzorčenjem iz velike žile pri evtanaziji živali.
Laboratories should use quality control methods, such as control charts (e.g. C-charts or X-bar charts (23)), to identify how variable their data are, and to show that the methodology is ‘under control’ in their laboratory.
Pri kostnem mozgu ali eritrocitih iz periferne krvi se lahko celice glede na metodo analize takoj supravitalno obarvajo (16) (17) (18) ali pa se pripravijo razmazi, ki se nato obarvajo za mikroskopijo ali fiksirajo in ustrezno obarvajo za analizo s pretočnim citometrom.
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 (24).
Uporaba DNK-specifičnega barvila [npr. akridin oranž (35) ali Hoechst 33258 plus pyronin-Y (36)] lahko prepreči nastajanje nekaterih artefaktov, povezanih z uporabo barvila, ki ni DNK-specifično.
Where the laboratory does not complete a sufficient number of experiments to establish a statistically robust negative control distribution (see paragraph 15) during the proficiency investigations (described in paragraph 13), it is acceptable that the distribution can be built during the first routine tests.
Kljub tej prednosti pa se lahko uporabijo običajna barvila (npr. Giemsa za mikroskopsko analizo).
This approach should follow the recommendations set out in the literature (24) and the negative control results obtained in these experiments should remain consistent with published negative control data.
Lahko se uporabijo tudi dodatni sistemi [npr. celulozne kolone za odstranitev celic z jedri (37) (38)], če je bilo dokazano, da so ti sistemi združljivi s pripravo vzorcev v laboratoriju.
Only major inconsistencies should result in the establishment of a new historical control database where expert judgement determines that it differs from the previous distribution (see paragraph 15).
Če se uporabljajo te metode, se lahko za opredelitev narave mikronukleusov (kromosom/fragment kromosoma) uporabijo antikinetohorna protitelesa (39), fluorescentna in situ hibridizacija (FISH) s pancentromernimi DNK-sondami (40) ali označevanje začetnih oligonukleotidov in situ s premazi, specifičnimi za centromere, skupaj z ustreznim nasprotnim barvanjem DNK (41), zato da se določi, ali mehanizem nastajanja mikronukleusov povzroča klastogena in/ali anevgena dejavnost.
Negative control data should consist of the incidence of micronucleated immature erythrocytes in each animal.
Za razlikovanje med anevgeni in klastogeni se lahko uporabijo druge metode, če so se izkazale za učinkovite.
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 15) and no evidence of technical or human failure.
Analiza (ročna in avtomatizirana)
Mice, rats, or another appropriate mammalian species may be used.
Vse mikroskopske preparate ali vzorce, vključno s tistimi s pozitivnimi in negativnimi kontrolami, je treba pred analizo neodvisno označiti in naključno razdeliti, tako da oseba, ki beleži ugotovitve, ne pozna pogojev tretiranja; če se uporablja avtomatiziran sistem štetja, ki se ne opira na vizualne preglede in nanj izvajalec ne more vplivati, tako označevanje ni potrebno.
When peripheral blood is used, it must be established that splenic removal of micronucleated cells from the circulation does not compromise the detection of induced micronuclei in the species selected.
Za vsako žival se določi delež nezrelih eritrocitov med vsemi (nezrelimi + zrelimi) eritrociti, tako da se prešteje skupno vsaj 500 eritrocitov pri kostnem mozgu in 2000 eritrocitov pri periferni krvi (42).
This has been clearly demonstrated for mouse and rat peripheral blood (2).
V določanje pojavnosti nezrelih eritrocitov z mikronukleusi je treba vključiti vsaj 4000 nezrelih eritrocitov na žival (43).
If species other than rodents are used, it is recommended that the measurement of induced micronuclei be integrated into another appropriate toxicity test.
Če je v zbirki podatkov o negativnih kontrolah iz preteklih preskusov v preskusnem laboratoriju srednja vrednost pogostosti nezrelih eritrocitov z mikronukleusi v ozadju < 0,1 %, je treba razmisliti o štetju dodatnih celic.
Test Conditions
Pri analiziranju vzorcev delež nezrelih eritrocitov med vsemi eritrociti v tretiranih živalih pri štetju z mikroskopijo ne sme biti nižji od 20 % deleža v kontroli z vehiklom/topilom, pri štetju CD71-pozitivnih nezrelih eritrocitov s citometričnimi metodami pa nižji od približno 5 % deleža v kontroli z vehiklom/topilom (glej odstavek 31) (29).
The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be capable of chemical reaction with the test chemicals.
Če je na primer pri preskusu kostnega mozga pri štetju z mikroskopijo kontrolni delež nezrelih eritrocitov v kostnem mozgu 50 %, je zgornja meja toksičnosti enaka 10 % nezrelih eritrocitov.
In the absence of historical or published control data showing that no micronuclei and other deleterious effects are induced by a chosen atypical solvent/vehicle, an initial study should be conducted in order to establish the acceptability of the solvent/vehicle control.
Ker vranica podgane zadrži in uniči eritrocite z mikronukleusi, je za vzdrževanje visoke občutljivosti poskusa pri analiziranju periferne krvi podgane analizo nezrelih eritrocitov z mikronukleusi bolje omejiti na najmlajšo frakcijo.
When a concurrent positive control group is not included, scoring controls (fixed and unstained slides or cell suspension samples, as appropriate for the method of scoring) should be included in each experiment.
Pri metodah za avtomatizirano analizo je mogoče te najbolj nezrele eritrocite opredeliti na podlagi njihove visoke vsebnosti RNK ali visoke ravni receptorjev transferina (CD71-pozitivni), izraženih na njihovi površini (31).
These can be obtained by including within the scoring of the study appropriate reference samples that have been obtained and stored from a separate positive control experiment conducted periodically (e.g. every 6-18 months); for example, during proficiency testing and on a regular basis thereafter, where necessary.
Vendar je neposredna primerjava različnih metod obarvanja pokazala, da je zadovoljive rezultate mogoče dobiti z različnimi metodami, vključno z običajnim barvanjem z barvilom akridin oranž (3) (4).
Positive control chemicals should reliably produce a detectable increase in micronucleus frequency over the spontaneous level.
Za vsako analizirano žival posebej je treba navesti število štetih nezrelih eritrocitov, število nezrelih eritrocitov z mikronukleusi in število nezrelih eritrocitov med vsemi eritrociti.
When employing manual scoring by microscopy, positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded samples to the scorer.
Kadar se miši tretirajo neprekinjeno 4 tedne ali več, je treba navesti tudi podatke o številu in deležu zrelih eritrocitov z mikronukleusi, če so zbrani.
Chemicals and CASRN
a. podatki o sočasni negativni kontroli se štejejo kot sprejemljivi za vključitev v zbirko podatkov laboratorija o kontrolah iz preteklih preskusov (glej odstavke 15 do 18);
Ethyl methanesulphonate [CASRN 62-50-0]
b. sočasne pozitivne kontrole ali kontrole za štetje morajo povzročiti odzive, skladne z odzivi iz zbirke podatkov o pozitivnih kontrolah iz preteklih preskusov, in statistično značilno povečanje v primerjavi s sočasno negativno kontrolo (glej odstavka 24 in 25);
Methyl methanesulphonate [CASRN 66-27-3]
c. analizirano je ustrezno število odmerkov in celic;
Ethyl nitrosourea [CASRN 759-73-9]
d. merila za izbor najvišjega odmerka so skladna z merili, opisanimi v odstavkih 30 do 33.
Mitomycin C [CASRN 50-07-7]
a. se pri vsaj eni od tretiranih skupin pokaže statistično značilno povečanje pogostosti nezrelih eritrocitov z mikronukleusi v primerjavi s sočasno negativno kontrolo;
Cyclophosphamide (monohydrate) [CASRN 50-18-0 (CASRN 6055-19-2)]
Priporočila glede najustreznejših statističnih metod so navedena v virih (44) (45) (46) (47).
Triethylenemelamine [CASRN 51-18-3]
Pozitivni rezultati pri preskusu mikronukleusov kažejo, da preskusna kemikalija povzroča nastanek mikronukleusov, ki so posledica poškodb kromosomov ali mitotičnega aparata v eritroblastih preskušane vrste.
Colchicine [CASRN 64-86-8] or Vinblastine [CASRN 865-21-4] — as aneugens
Če je preskus izveden za odkrivanje centromer v mikronukleusih, je preskusna kemikalija, ki povzroča nastanek mikronukleusov s centromerami (centromerne DNK ali kinetohora, ki so znak izgube celih kromosomov), dokaz, da je preskusna kemikalija anevgen.
However, if consistent inter-animal variability and frequencies of cells with micronuclei are demonstrated by historical negative control data at each sampling time for the testing laboratory, only a single sampling for the negative control may be necessary.
Pod pogojem, da so izpolnjena vsa merila za sprejemljivost, se preskusna kemikalija šteje za jasno negativno, če se v katerem koli od proučevanih preskusnih pogojev:
If peripheral blood is used, a pre-treatment sample is acceptable instead of a concurrent negative control for short-term studies when the resulting data are consistent with the historical control database for the testing laboratory.
a. pri nobeni tretirani skupini ne pokaže statistično značilno povečanje pogostosti nezrelih eritrocitov z mikronukleusi v primerjavi s sočasno negativno kontrolo;
It has been shown for rats that pre-treatment sampling of small volumes (e.g. below 100 μl/day) has minimal impact on micronucleus background frequency (25).
Dokazi o izpostavljenosti kostnega mozga preskusni kemikaliji lahko vključujejo zmanjšanje razmerja nezrelih eritrocitov ali meritve ravni preskusne kemikalije v plazmi ali krvi.
In general, the micronucleus response is similar between male and female animals and, therefore, most studies could be performed in either sex (26).
Pri intravenoznem dajanju dokaz o izpostavljenosti ni potreben.
Data demonstrating relevant differences between males and females (e.g. differences in systemic toxicity, metabolism, bioavailability, bone marrow toxicity, etc. including e.g. in a range-finding study) would encourage the use of both sexes.
vir, številka serije, rok uporabe, če je na voljo;
As a guide to maximum typical animal requirements, a study in bone marrow conducted according to the parameters established in paragraph 37 with three dose groups and concurrent negative and positive controls (each group composed of five animals of a single sex) would require between 25 and 35 animals.
topnost in stabilnost preskusne kemikalije v topilu/vehiklu, če je znana;
If a preliminary range-finding study is performed because there are no suitable data already available to aid in dose selection, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (27).
podatki o pozitivnih in negativnih kontrolah (z vehiklom/topilom);
The study should aim to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia (28)).
metode za preverjanje, ali je preskusna kemikalija dosegla krvni obtok ali ciljno tkivo;
The highest dose may also be defined as a dose that produces toxicity in the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood of more than 50 %, but to not less than 20 % of the control value).
merila za štetje nezrelih eritrocitov z mikronukleusi;
However, when analysing CD71-positive cells in peripheral blood circulation (i.e., by flow cytometry), this very young fraction of immature erythrocytes responds to toxic challenges more quickly than the larger RNA-positive cohort of immature erythrocytes.
število analiziranih celic na žival pri določanju pogostosti nezrelih eritrocitov z mikronukleusi in za določanje deleža nezrelih eritrocitov glede na zrele;
Therefore, higher apparent toxicity may be evident with acute exposure designs examining the CD71-positive immature erythrocyte fraction as compared to those that identify immature erythrocytes based on RNA content.
metode, kot so uporaba antikinetohornih protiteles ali centromernih DNK-sond, da se ugotovi, ali mikronukleusi vsebujejo cele kromosome ali njihove fragmente, če je ustrezno.
For this reason, when experiments utilise five or fewer days of treatment, the highest dose level for test chemicals causing toxicity may be defined as the dose that causes a statistically significant reduction in the proportion of CD71-positive immature erythrocytes among total erythrocytes but not to less than 5 % of the control value (29).
delež nezrelih eritrocitov med vsemi eritrociti;
Chemicals that exhibit saturation of toxicokinetic properties, or induce detoxification processes that may lead to a decrease in exposure after long-term administration may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis.
število nezrelih eritrocitov z mikronukleusi, navedeno za vsako žival posebej;
If the test chemical does not produce toxicity in a range-finding study or based on existing data, the highest dose for an administration period of 14 days or more should be 1000 mg/kg body weight/day, or for administration periods of less than 14 days, 2000 mg/kg/body weight/day.
srednja vrednost ± standardni odklon nezrelih eritrocitov z mikronukleusi na skupino;
When administration occurs for 14 days or more, the limit dose is 1000 mg/kg body weight/day.
uporabljene statistične analize in metode;
For administration periods of less than 14 days, the limit dose is 2000 mg/kg/body weight/day.
podatki o sočasnih negativnih in pozitivnih kontrolah z območji, srednjimi vrednostmi in standardnimi odkloni;
Therefore, routes of exposure such as dietary, drinking water, topical subcutaneous, intravenous, oral (by gavage), inhalation, intratracheal, or implantation may be chosen as justified.
podatki o negativnih in pozitivnih kontrolah iz preteklih preskusov z območji, srednjimi vrednostmi in standardnimi odkloni in 95-odstotne kontrolne meje za porazdelitev ter obravnavano časovno obdobje in število opazovanj;