natgillin/translations-raw huggingface dataset:
Den relevante kolonne installeres i HPLC-systemet.
A murine test, the Local Lymph Node Assay (LLNA, TM B.42 (4)) and its two non-radioactive modifications, LLNA: DA (TM B.50 (5)) and LLNA: BrdU-ELISA (TM B.51 (6)), which all assess the induction response exclusively, have also gained acceptance since they provide an advantage over the guinea pig tests in terms of animal welfare and an objective measurement of the induction phase of skin sensitisation.
Den HPLC-opstilling, der er beskrevet i den validerede protokol, består af en Zorbax SB-C-18 2,1 mm × 100 mm × 3,5 mμ som foretrukken kolonne.
More recently, mechanistically based in chemico and in vitro test methods have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals.
Med denne HPLC-kolonne med omvendt fase bør hele systemet bringes i ligevægt ved 30 °C med 50 % fase A (0,1 % (v/v) trifluoreddikesyre i vand) og 50 % fase B (0,085 % (v/v) trifluoreddikesyre i acetonitril) i mindst 2 timer før kørsel.
However, combinations of non-animal methods (in silico, in chemico, in vitro) within Integrated Approaches to Testing and Assessment (IATA) will be needed to be able to fully substitute for the animal tests currently in use given the restricted AOP mechanistic coverage of each of the currently available non-animal test methods (2) (7).
HPLC-analysen bør udføres ved en flowhastighed på 0,35 ml/min. og en lineær gradient fra 10 % til 25 % acetonitril i 10 minutter efterfulgt af en hurtig stigning til 90 % acetonitril for at fjerne andre materialer.
The DPRA is proposed to address the molecular initiating event of the skin sensitisation AOP, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine (8).
Der bør indsprøjtes lige store mængder af hver enkelt standard, prøve og kontrol.
Cysteine and lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers (9).
Kolonnen bør bringes i ligevægt igen under de oprindelige forhold i 7 minutter mellem injektionerne.
The DPRA has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid (10) to be used as part of an IATA to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Hvis der anvendes en anden HPLC-kolonne med omvendt fase, kan det være nødvendigt at justere de indstillingsparametre, der er beskrevet ovenfor, for at sikre en passende eluering og integration af cystein- og lysinpeptiderne, herunder injektionsvolumen, der kan variere alt efter det anvendte system (typisk i størrelsesordenen 3-10 μl).
Examples on the use of DPRA data in combination with other information are reported in the literature (11) (12) (13) (14).
Hvis der anvendes en alternativ HPLC-opstilling, er det vigtigt at dokumentere dens ækvivalens med den validerede opsætning, som er beskrevet ovenfor (f.eks. ved at teste kompetencestofferne i tillæg 2).
Definitions are provided in Appendix I.
Absorbans måles ved 220 nm.
INITIAL CONSIDERATIONS, APPLICABILITY AND LIMITATIONS
Hvis en fotodiodearraydetektor anvendes, registreres absorbansen ved 258 nm ligeledes.
The correlation of protein reactivity with skin sensitisation potential is well established (15) (16) (17).
Det skal bemærkes, at nogle leverancer af acetonitril kan have en negativ indvirkning på peptidstabiliteten, og dette skal vurderes, når der anvendes et nyt batch acetonitril.
Nevertheless, since protein binding represents only one key event, albeit the molecular initiating event of the skin sensitisation AOP, protein reactivity information generated with testing and non-testing methods may not be sufficient on its own to conclude on the absence of skin sensitisation potential of chemicals.
Forholdet mellem toparealet ved 220 nm og toparealet ved 258 nm kan bruges som indikator for co-elueringen.
Therefore, data generated with this test method should be considered in the context of integrated approaches such as IATA, combining them with other complementary information e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods including read-across from chemical analogues.
For hver prøve vil et forhold i området 90 % < gennemsnitligt [13] områdeforhold for kontrolprøver < 100 % være en god indikation af, at co-elueringen ikke har fundet sted.
This test method can be used, in combination with other complementary information, to support the discrimination between skin sensitisers (i.e. UN GHS/CLP Category 1) and non-sensitisers in the context of IATA.
Der kan være testkemikalier, som kan fremme oxideringen af cysteinpeptidet.
This test method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN GHS/CLP, nor to predict potency for safety assessment decisions.
Topniveauet for det dimeriserede cysteinpeptid kan kontrolleres visuelt.
However, depending on the regulatory framework, a positive result with the DPRA may be used on its own to classify a chemical into UN GHS/CLP category 1.
Hvis dimeriseringen tilsyneladende er indtruffet, bør dette noteres, eftersom den procentvise peptidnedbrydning kan overvurderes, hvilket kan føre til falsk positive forudsigelser og/eller placering i en højere reaktivitetsklasse (jf. punkt 29 og 30).
The DPRA test method proved to be transferable to laboratories experienced in high-performance liquid chromatography (HPLC) analysis.
HPLC-analyse af cystein- og lysinpeptider kan udføres samtidig (hvis der er to HPLC-systemer til rådighed) eller på forskellige dage.
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).
Hvis analysen udføres på forskellige dage, bør alle kemiske opløsninger fremstilles for begge assays på hver af disse dage.
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.
Tidspunktet for analysen vælges således, at injektionen af den første prøve indledes 22-26 timer efter, at testkemikaliet blev blandet med peptidopløsningen.
1) from non-sensitisers is 80 % (N=157) with a sensitivity of 80 % (88/109) and specificity of 77 % (37/48) when compared to LLNA results.
Til den HPLC-opstilling, der blev brugt i valideringsundersøgelsen, og som beskrives i denne forsøgsmetode, kan der medtages op til 26 analyseprøver i en enkelt HPLC-kørsel (se også punkt 17).
The DPRA is more likely to under predict chemicals showing a low to moderate skin sensitisation potency (i.e. UN GHS/CLP subcategory 1B) than chemicals showing a high skin sensitisation potency (i.e. UN GHS/CLP subcategory 1A) (18) (19).
Tillæg 3 indeholder et eksempel på HPLC-analysesekvensen.
However, the accuracy values given here for the DPRA as a stand-alone test method are only indicative since the test method should be considered in combination with other sources of information in the context of an IATA and in accordance with the provisions of paragraph 9 above.
Dataevaluering
Furthermore when evaluating non-animal methods for skin sensitisation, it should be kept in mind that the LLNA test as well as other animal tests may not fully reflect the situation in the species of interest, i.e. humans.
Koncentrationen af cystein-eller lysinpeptidet bestemmes fotometrisk ved 220 nm i hver prøve ved at måle toparealet (areal under kurven, AUC) for de relevante toppe og ved at beregne peptidkoncentrationen ved hjælp af den lineære kalibreringskurve, som er afledt af disse standarder.
On the basis of the overall data available, the DPRA was shown to be applicable to test chemicals covering a variety of organic functional groups, reaction mechanisms, skin sensitisation potency (as determined in in vivo studies) and physico-chemical properties (8) (9) (10) (19).
Den procentvise peptidnedbrydning bestemmes i hver prøve ved at måle toparealet og dividere det med det gennemsnitlige topareal for de relevante referencekontroller C (se tillæg 3) i henhold til nedenstående formel.
Taken together, this information indicates the usefulness of the DPRA to contribute to the identification of skin sensitisation hazard.
Peptidnedbrydning
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.
Peptidtopareal i replikat injektion
This test method is not applicable for the testing of metal compounds since they are known to react with proteins with mechanisms other than covalent binding.
Gnsn.peptidtopareal i referencekontroller C
A test chemical should be soluble in an appropriate solvent at a final concentration of 100 mM (see paragraph 18).
Acceptkriterier
However, test chemicals that are not soluble at this concentration may still be tested at lower soluble concentrations.
Følgende kriterier bør være opfyldt, for at en kørsel kan anses for gyldig:
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.
Standardkalibreringskurven bør have en r2 > 0,99.
Limited information is currently available on the applicability of the DPRA to mixtures of known composition (18) (19).
Den gennemsnitlige peptidnedbrydningsværdi i procent for de tre replikater for kanelaldehyden til den positive kontrol bør være mellem 60,8 % og 100 % for cysteinpeptidet og mellem 40,2 % og 69,0 % for lysinpeptidet, og den maksimale standardafvigelse for de positive kontrolreplikater bør være < 14,9 % for den procentvise cysteinnedbrydning og < 11,6 % for den procentvise lysinnedbrydning.
The DPRA is nevertheless considered to be technically applicable to the testing of multi-constituent substances and mixtures of known composition (see paragraph 18).
Den gennemsnitlige peptidkoncentration for referencekontrol A bør være 0,5 ± 0,05 mM og variationskoefficienten for peptidernes toparealer for de ni referencekontroller B og C i acetonitril bør være < 15,0 %.
Before use of this test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose.
Hvis et eller flere af disse kriterier ikke er opfyldt, bør kørslen gentages.
Such considerations are not needed when there is a regulatory requirement for testing of the mixture.
Følgende kriterier bør være opfyldt, hvis testresultatet for kemikaliet skal anses for gyldigt:
The current prediction model cannot be used for complex mixtures of unknown composition or for substances of unknown or variable composition, complex reaction products or biological materials (i.e. UVCB substances) due to the defined molar ratio of test chemical and peptide.
Den maksimale standardafvigelse for replikaterne af testkemikaliet bør være < 14,9 % for den procentvise cysteinnedbrydning og < 11,6 % for den procentvise lysinnedbrydning,
For this purpose a new prediction model based on a gravimetric approach will need to be developed.
Den gennemsnitlige peptidkoncentration af de tre referencekontroller C i det relevante opløsningsmiddel bør være 0,50 ± 0,05 mM.
In cases where evidence can be demonstrated on the non-applicability of the test method to other specific categories of chemicals, the test method should not be used for those specific categories of chemicals.
Hvis disse kriterier ikke er opfyldt, bør dataene afvises og kørslen gentages for det pågældende testkemikalie.
This test method is an in chemico method that does not encompass a metabolic system.
Forudsigelsesmodel
Chemicals that require enzymatic bioactivation to exert their skin sensitisation potential (i.e. pro-haptens) cannot be detected by the test method.
Den gennemsnitlige procentvise cystein- og lysinnedbrydningsværdi i procent beregnes for hvert enkelt testkemikalie.
Chemicals that become sensitisers after abiotic transformation (i.e. pre-haptens) are reported to be in some cases correctly detected by the test method (18).
Negativ nedbrydning betragtes som “0” ved beregning af middelværdien.
In the light of the above, negative results obtained with the test method should be interpreted in the context of the stated limitations and in the connection with other information sources within the framework of an IATA.
Ved brug af cystein 1:10-/lysin 1:50-forudsigelsesmodellen i tabel 1 bør grænsen på 6,38 % for den gennemsnitlige peptidnedbrydning anvendes til at underbygge sondringen mellem hudsensibiliserende og ikke-hudsensibiliserende stoffer inden for rammerne af en IATA.
Test chemicals that do not covalently bind to the peptide but promote its oxidation (i.e. cysteine dimerisation) could lead to a potential over estimation of peptide depletion, resulting in possible false positive predictions and/or assignement to a higher reactivity class (see paragraphs 29 and 30).
Det kan måske være nyttigt at anvende forudsigelsesmodellen til at placere et testkemikalie i en reaktivitetsklasse (dvs. lav, moderat og høj reaktivitet) som grundlag for styrkevurderingen inden for rammerne af en IATA.
As described, the DPRA supports the discrimination between skin sensitisers and non-sensitisers.
Cystein 1:10/lysin 1:50-forudsigelsesmodel [14]
However, it may also potentially contribute to the assessment of sensitising potency (11) when used in integrated approaches such as IATA.
Middelværdi for procentvis cystein- og lysinnedbrydning
However further work, preferably based on human data, is required to determine how DPRA results may possibly inform potency assessment.
Reaktivitetsklasse
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test chemical at 25 ± 2,5 °C.
DPRA-forudsigelse [15]
The synthetic peptides contain phenylalanine to aid in the detection.
0 % < middelværdi for procentvis nedbrydning ≤ 6,38 %
Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.
Ingen eller minimal reaktivitet
Cysteine- and lysine peptide percent depletion values are then calculated and used in a prediction model (see paragraph 29) which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
Negativ
Prior to routine use of the method described in this test method, laboratories should demonstrate technical proficiency, using the ten proficiency substances listed in Appendix 2.
6,38 % < middelværdi for procentvis nedbrydning ≤ 22,62 %
This test method is based on the DPRA DB-ALM protocol no 154 (20) which represents the protocol used for the EURL ECVAM-coordinated validation study.
Lav reaktivitet
It is recommended that this protocol is used when implementing and using the method in the laboratory.
Positiv
The following is a description of the main components and procedures for the DPRA.
22,62 % < middelværdi for procentvis nedbrydning ≤ 42,47 %
If an alternative HPLC set-up is used, its equivalence to the validated set-up described in the DB-ALM protocol should be demonstrated (e.g. by testing the proficiency substances in Appendix 2).
Moderat reaktivitet
Preparation of the cysteine or lysine-containing peptides
42,47 % < middelværdi for procentvis nedbrydning ≤ 100 %
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 85 % and preferably in the range of 90-95 %, should be freshly prepared just before their incubation with the test chemical.
Høj reaktivitet
The final concentration of the cysteine peptide should be 0,667 mM in pH 7,5 phosphate buffer whereas the final concentration of the lysine peptide should be 0,667 mM in pH 10,2 ammonium acetate buffer.
Der kan være tilfælde, hvor testkemikaliet (stoffet eller en eller flere bestanddele af et stof eller en blanding) absorberer betydeligt ved 220 nm og har samme retentionstid for peptidet (co-eluering).
The HPLC run sequence should be set up in order to keep the HPLC analysis time less than 30 hours.
Co-eluering kan afhjælpes ved at justere HPLC-opstillingen smule med henblik på at adskille elueringstiden for testkemikaliet og peptidet yderligere.
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.
Hvis der anvendes en alternativ HPLC-opstilling for at forsøge at afhjælpe co-elueringen, bør man dokumentere dens ækvivalens med den validerede opstilling (f.eks. ved at teste kompetencestofferne i tillæg 2).
All of the replicates analysed in the same run should use the identical cysteine and lysine peptide stock solutions.
Når co-eluering forekommer, kan topområdet for peptidet ikke kan integreres, og det er ikke muligt at beregne den procentvise nedbrydning af peptidet.
It is recommended to prove individual peptide batches for proper solubility prior to their use.
Hvis co-eluering af sådanne testkemikalier forekommer i forbindelse med både cystein- og lysinpeptider, bør analysen rapporteres som “usikker”.
Preparation of the test chemical
I tilfælde, hvor co-eluering kun forekommer med lysinpeptidet, kan cystein 1:10-modellen, som er beskrevet i tabel 2, anvendes.
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).
Cystein 1:10-forudsigelsesmodel [16]
An appropriate solvent will dissolve the test chemical completely.
Procentvis cystein- (Cys) nedbrydning
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.
Reaktivitetsklasse
Suitable solvents are acetonitrile, water, 1:1 mixture water:acetonitrile, isopropanol, acetone or 1:1 mixture acetone:acetonitrile.
DPRA-forudsigelse [17]
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).
0 % ≤ procentvis Cys-nedbrydning ≤ 13,89 %
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.
13,89 % < procentvis Cys-nedbrydning ≤ 23,09 %
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.
23,09 % < procentvis Cys-nedbrydning ≤ 98,24 %
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.
98,24 % < procentvis Cys-nedbrydning ≤ 100 %
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.
Der kan være andre tilfælde, hvor overlappet i retentionstid mellem testkemikaliet og de to peptider er ufuldstændig.
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.
I sådanne tilfælde kan man vurdere de procentvise peptidnedbrydningsværdier og anvende dem i cystein 1:10-/lysin 1:50-modellen, men det er imidlertid ikke muligt at foretage en præcis placering af testkemikaliet i en reaktivitetsklasse.
However, when testing mixtures, multi-constituent substances or polymers of known composition, it should be considered to also test the neat chemical.
En enkelt HPLC-analyse for både cystein- og lysinpeptidet bør være tilstrækkelig for et testkemikalie, når resultatet er entydigt.
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.
Men i tilfælde af resultater, der ligger tæt på den tærskelværdi, der bruges til at skelne mellem positive og negative resultater (dvs. usikre resultater), kan der være behov for yderligere undersøgelser.
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.
Hvis situationer, hvor den gennemsnitlige procentvise nedbrydning ligger i området 3 % til 10 % for cystein 1:10-/lysin 1:50-forudsigelsesmodellen, eller den procentvise cysteinnedbrydning ligger i området 9 %-17 % for cystein 1:10-forudsigelsesmodellen, bør man overveje endnu en kørsel og en tredje kørsel, hvis de to første kørsler giver modstridende resultater.
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.
Testrapporten bør indeholde følgende oplysninger
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.
Stof med kun en bestanddel
Preparation of the positive control, reference controls and coelution controls
kemisk identifikation, f.eks. IUPAC- eller CAS-navn(e), CAS-nummer/numre, SMILES- eller InChI-kode, strukturformel og/eller andre identifikatorer
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.
fysisk udseende, vandopløselighed, molekylvægt og yderligere relevante fysisk-kemiske egenskaber i den udstrækning, det er muligt
Other suitable positive controls preferentially providing mid-range depletion values may be used if historical data are available to derive comparable run acceptance criteria.
renhed, kemisk identitet af urenheder i det omfang, det er relevant og praktisk muligt osv.
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).
eventuel behandling inden testen (f.eks. opvarmning, formaling)
The appropriate reference control for each chemical is used to calculate the percent peptide depletion for that chemical (see paragraph 26).
testede koncentrationer
In addition a co-elution control constituted by the test chemical alone for each of the test chemicals analysed should be included in the run sequence to detect possible co-elution of the test chemical with either the lysine or the cysteine peptide.
opbevaring og stabilitet i det omfang, det er muligt.
Incubation of the test chemical with the cysteine and lysine peptide solutions