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Z hodnôt pH a absorbancie pri zvolenej vlnovej dĺžke sa vypočíta dostatočný počet hodnôt pKa, pričom sa použije aspoň 5 hodnôt pH, pri ktorých je látka ionizovaná najmenej na 10 percent a zároveň menej ako na 90 percent.
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Okrem toho by sa pri hodnotení metód na zistenie kožnej senzibilizácie bez použitia zvierat malo zohľadniť to, že test LLNA ani iné testy na zvieratách nemusia v plnej miere odzrkadľovať situáciu u druhov, ktoré sú predmetom záujmu, čiže u ľudí.
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Na základe celkových dostupných údajov sa preukázalo, že testovaciu metódu DPRA možno použiť v prípade testovaných chemikálií, ktoré predstavujú rôzne organické funkčné skupiny, majú rôzne mechanizmy reakcie, potenciál spôsobiť kožnú senzibilizáciu (ako bolo stanovené v štúdiách in vivo) a rôzne fyzikálno-chemické vlastnosti (8) (9) (10) (19).
Elhajouji, A., E. Lorge (2006).
Celkovo z týchto informácií vyplýva, že testovacia metóda DPRA je užitočná a môže prispieť k zisteniu nebezpečnosti kožnej senzibilizácie.
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Termín „testovaná chemikália“ sa v tejto testovacej metóde používa na označenie toho, čo sa testuje, a nesúvisí s použiteľnosťou DPRA na testovanie látok a/nebo zmesí.
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Túto testovaciu metódu nemožno použiť na testovanie zlúčenín kovov, keďže je o nich známe, že reagujú s proteínmi prostredníctvom mechanizmov iných než kovalentnou väzbou.
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Ihneď po odstránení testovaných a kontrolných chemikálií sa do inzertov (napr. Millicell-HA) pridá 400 μl z 0,1 mg/ml roztoku nátrium-fluoresceínu [0,01 hm. % HBSS s obsahom vápnika (pri koncentrácii 1,0 – 1,8 mM) a bez fenolovej červene].
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Na konci inkubácie fluoresceínom sa inzerty opatrne vyberú z každej jamky.
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Vykoná sa vizuálna kontrola všetkých filtrov a zaznamená sa každé poškodenie, ku ktorému mohlo dôjsť pri manipulácii.
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V roztoku, ktorý zostal v jamkách po odstránení inzertov, sa vyčísli množstvo fluoresceínu, ktoré uniklo cez monovrstvu a inzert.
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V prípade použitia iných druhov čeľade dafniovité by sa mal daný druh jasne identifikovať a jeho použitie opodstatniť.
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Hodnota pH by sa mala pohybovať v rozmedzí 6,0 až 8,5 na začiatku testu, no počas príslušného testu by nemala kolísať o viac ako ± 0,5 jednotiek pH.
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Aby voda na riedenie neovplyvňovala neprimeraným spôsobom výsledky testovania (napríklad vytváraním komplexov s testovanou látkou) alebo aby nemala nepriaznivý vplyv na stav násady rýb, je potrebné zabezpečiť v pravidelných intervaloch – aspoň na začiatku a konci testu – odber vzoriek na analýzu.
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To znamená, že krátko pred prvým podaním potravy bez pridanej testovanej látky by sa mala odobrať finálna vzorka z vychytávacej fázy a prvá vzorka z depuračnej fázy by sa mala odobrať približne 23 hodín po prvom podaní krmiva pridanej testovanej látky.
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The level of reproducibility in predictions that can be expected from the test method is in the order of 85 % within laboratories and 80 % between laboratories (10).
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Results generated in the validation study (18) and published studies (19) overall indicate that the accuracy of the DPRA in discriminating sensitisers (i.e. UN GHS/CLP Cat.
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The term “test chemical” is used in this test method to refer to what is being tested and is not related to the applicability of the DPRA to the testing of substances and/or mixtures.
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A test chemical should be soluble in an appropriate solvent at a final concentration of 100 mM (see paragraph 18).
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In such a case, a positive result could still be used to support the identification of the test chemical as a skin sensitiser but no firm conclusion on the lack of reactivity should be drawn from a negative result.
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Reliability
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The proportion of all negative/inactive chemicals that are correctly classified by the test method.
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It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method (21).
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80-100
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0-7
inherentná vlastnosť činidla, prípadne situácia, ktorá môže vyvolať nepriaznivé účinky pri expozícii organizmu, systému alebo (sub)populácie účinkom daného činidla.
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0-25
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10-45
Po expozícii trvajúcej 1 minútu pri izbovej teplote by sa testovaná chemikália mala opatrne odstrániť odsatím, monovrstva by sa mala dvakrát premyť sterilným, teplým (37 °C) HBSS s obsahom vápnika (pri koncentrácii 1,0 – 1,8 mM) a bez fenolovej červene a ihneď potom sa zmeria únik fluoresceínu.
0-5,5
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