OECD 442 Flashcards
(146 cards)
1
Q
What is the LLNA: DA?
A
A modified, nonradioactive version of the LLNA for identifying skin sensitizers
2
Q
What is the primary advantage of LLNA: DA over TG 406?
A
Reduces animal use and minimizes pain by avoiding dermal hypersensitivity reactions
3
Q
What does TG 429 use to measure lymphocyte proliferation?
A
Radioisotopic thymidine/iodine
4
Q
What method does TG 442A use to measure lymphocyte proliferation?
A
Adenosine triphosphate (ATP) bioluminescence
5
Q
What are the limitations of LLNA: DA?
A
Not suitable for certain metals, skin irritants, and some substances affecting ATP levels
6
Q
What does an SI ≥ 1.8 suggest in the LLNA: DA?
A
Potential skin sensitization
7
Q
What is the principle behind the LLNA: DA method?
A
Detects skin sensitizers by measuring lymphocyte proliferation in lymph nodes
8
Q
What is measured to determine lymphocyte proliferation in LLNA: DA?
A
ATP content in lymphocytes via bioluminescence
9
Q
What are the key procedural steps in the LLNA: DA assay?
A
Animal selection, housing & feeding, animal preparation, dosing solutions, reliability check
10
Q
What is the preferred positive control for LLNA: DA?
A
25% hexyl cinnamic aldehyde or 25% eugenol in acetone: olive oil (4:1)
11
Q
What is the purpose of the prescreen test in LLNA: DA?
A
Determines the maximum dose for the main study when toxicity data is unavailable
12
Q
Fill in the blank: The LLNA: DA method uses _______ for measuring lymphocyte proliferation.
A
adenosine triphosphate (ATP) bioluminescence
13
Q
What is the significance of the Stimulation Index (SI) in LLNA: DA?
A
Indicates the degree of skin sensitization potential
14
Q
True or False: The LLNA: DA can replace all traditional guinea pig tests.
A
False
15
Q
What does the Adverse Outcome Pathway (AOP) for skin sensitization describe?
A
Key events from chemical structure to in vivo outcome
16
Q
What is the role of BrdU in the LLNA: BrdUELISA method?
A
A thymidine analogue incorporated into proliferating cells
17
Q
What type of mice is preferred for the LLNA: BrdUELISA test?
A
Young adult female CBA/JN mice
18
Q
What is the main purpose of the LLNA: BrdUELISA?
A
Identifying skin sensitizing chemicals
19
Q
What does an SI value of ≥ 1.6 indicate in LLNA: BrdUELISA?
A
Potential skin sensitizer
20
Q
What does the LLNA: BrdUFCM method measure?
A
Lymphocyte proliferation in lymph nodes
21
Q
What are benchmark substances used for in LLNA?
A
Evaluating specific chemical classes
22
Q
What common vehicles are used to dissolve test substances in LLNA?
A
Acetone: olive oil, DMF, DMSO
23
Q
What should be monitored daily during the LLNA study?
A
Signs of local irritation or systemic toxicity
24
Q
What is the significance of statistical analysis in LLNA results?
A
Assesses dose-response trends and group comparisons
25
What is the main goal of the OECD/OCDE 442A and 442B guidelines?
Support animal testing for skin sensitization while reducing animal use
26
What is the LLNA: BrdUFCM?
A validated, nonradioactive alternative to traditional skin sensitization tests
## Footnote
It reduces animal use and distress, but has limitations for certain substances.
27
What does an SI ≥ 2.7 indicate in the LLNA: BrdUFCM?
A potential skin sensitizer
## Footnote
SI stands for Stimulation Index.
28
What is the main principle of the LLNA: BrdUFCM test?
It measures lymphocyte proliferation in lymph nodes by analyzing BrdU incorporation into DNA.
29
What types of mice are used in the LLNA: BrdUFCM test?
BALB/c or CBA/J mice.
30
How is the test chemical applied in the LLNA: BrdUFCM?
Topically to the ear.
31
What is included to ensure the accuracy of the LLNA: BrdUFCM test?
Positive controls (PC).
32
How many animals are required per dose group in the LLNA: BrdUFCM?
At least 4 animals per dose group.
33
What is the purpose of a prescreen test in the LLNA: BrdUFCM?
To determine the appropriate highest dose to use.
34
What is the first step in the main study experimental schedule of the LLNA: BrdUFCM?
Apply test chemical (25 μL) to each ear on Day 1.
35
What is the purpose of measuring BrdU-positive lymphocytes?
To assess cell proliferation using flow cytometry.
36
What statistical methods are used in the LLNA: BrdUFCM?
Linear regression and Dunnett’s test.
37
What does the Direct Peptide Reactivity Assay (DPRA) assess?
Covalent binding to proteins in skin sensitization.
38
What are the four classes of substances in the DPRA based on peptide depletion values?
Sensitizers, moderate sensitizers, weak sensitizers, and nonsensitizers.
39
What is the accuracy of the DPRA in discriminating between sensitizers and nonsensitizers?
80% accuracy.
40
What is a limitation of the DPRA?
It underpredicts substances with moderate skin sensitization potential.
41
What type of chemicals is the DPRA not applicable for?
Metal compounds or chemicals that require enzymatic bioactivation.
42
What is the principle of the DPRA test?
Measures peptide depletion after incubation with a test chemical.
43
What is the purpose of the Integrated Approaches to Testing and Assessment (IATA)?
To integrate and weight all relevant data for hazard identification and safety assessment.
44
What is the role of the kDPRA?
It distinguishes UN GHS subcategory 1A skin sensitizers from nonsubcategory 1A sensitizers.
45
What is required for HPLC calibration in the DPRA?
A standard calibration curve must be generated.
46
What is the acceptance criteria for a valid run in the DPRA?
The standard calibration curve must have an r² > 0.99.
47
What are common vehicles used in the LLNA: BrdUFCM for chemical preparation?
* Acetone: olive oil (4:1)* N,N-dimethylformamide.
48
What is the significance of the Stimulation Index (SI) in the LLNA: BrdUFCM?
It indicates the degree of lymphocyte proliferation in response to the test chemical.
49
What are the clinical observations recorded during the LLNA: BrdUFCM?
Signs of irritation or toxicity.
50
What is the purpose of positive control in the LLNA: BrdUFCM?
To ensure the test's accuracy.
51
What does the term 'in chemico' refer to in the context of skin sensitization testing?
It relates to assays that study covalent binding to proteins.
52
What is the method for measuring peptide concentration in the evaluation?
Peptide concentration is measured photometrically at 220 nm by analyzing the peak area (AUC) and using a calibration curve.
53
How is peptide depletion calculated?
Peptide depletion is calculated by comparing the peak area of the sample to the mean peak area of reference controls (C) using a specific formula.
54
What is the acceptance criterion for a valid standard calibration curve?
The standard calibration curve must have an r² > 0.99.
55
What are the acceptable depletion ranges for cysteine and lysine peptides using positive control?
* Cysteine peptide depletion: 60.8% - 100%
* Lysine peptide depletion: 40.2% - 69.0%
56
What is the maximum standard deviation allowed for replicates in cysteine and lysine?
* Cysteine: < 14.9%
* Lysine: < 11.6%
57
What is the mean peptide concentration requirement for reference controls A?
Reference controls A must have a mean peptide concentration of 0.50 ± 0.05 mM.
58
What is the coefficient of variation (CV) criterion for peptide peak areas in reference controls B and C?
The CV should be < 15.0%.
59
What happens if any acceptance criteria are not met?
The run should be repeated.
60
What is the threshold percentage for average peptide depletion in the prediction model?
The threshold is 6.38% average peptide depletion.
61
What is the significance of the prediction model in skin sensitization?
It helps distinguish between skin sensitisers and nonsensitisers and can assign a test chemical to a reactivity class.
62
What should be done if coelution occurs with a test chemical?
Adjust the HPLC setup to separate their elution times.
63
How is inconclusive analysis reported if coelution prevents peak integration?
The analysis should be reported as 'inconclusive'.
64
What is the recommended action for results near the threshold?
Additional testing is recommended.
65
What is the original threshold for a positive classification?
The original threshold is 6.38%.
66
What does ADRA stand for?
Amino Acid Derivative Reactivity Assay.
67
What is the purpose of the ADRA?
ADRA quantifies the reactivity of test chemicals toward synthetic amino acid derivatives to distinguish between skin sensitisers and nonsensitisers.
68
What validation accuracy does the ADRA show based on LLNA data?
ADRA shows an accuracy of 76% based on LLNA data.
69
What is the role of fluorescence detection in ADRA?
Fluorescence detection reduces the occurrence of coelution compared to UV detection.
70
What is the acceptable purity for NAC/NAL in ADRA?
Purity must be at least 98%.
71
What is the incubation condition for NAC/NAL in the ADRA test?
Incubate at 25°C for 24 hours in the dark.
72
What is the significance of the log kmax in kDPRA?
It distinguishes strong skin sensitizers from weaker ones or nonsensitizers.
73
What is the balanced accuracy of kDPRA compared to LLNA?
kDPRA shows 85% balanced accuracy.
74
What types of chemicals are not suitable for kDPRA?
* Metal compounds
* Lysinereactive chemicals
* Prohaptens requiring enzymatic activation
75
What are the testing considerations for kDPRA?
The test chemical must be soluble at 20 mM, and controls must be included to address fluorescence issues.
76
What is the main principle of the kDPRA test?
It quantifies the reactivity of test chemicals with a synthetic cysteine peptide.
77
What is the purpose of the kDPRA?
The kDPRA quantifies the reactivity of test chemicals with a cysteine peptide to assess skin sensitization
## Footnote
It is a modified version of the Direct Peptide Reactivity Assay (DPRA)
78
What are the key features of the kDPRA?
* Uses the same cysteine peptide (AcRFAACAACOOH) as DPRA
* Reaction medium is 25% acetonitrile in phosphate buffer
* Measures peptide depletion at five concentrations and six time points
* Conducted at 25±2.5ºC
79
What is the significance of a peptide depletion exceeding 13.89%?
If peptide depletion exceeds 13.89%, further calculations proceed
## Footnote
This indicates significant reactivity of the test chemical.
80
What does log kmax ≥ 2.0 indicate?
It indicates the test chemical is a UN GHS subcategory 1A skin sensitizer.
81
What are the controls required in the kDPRA?
* Positive Control: Cinnamic aldehyde at 20 mM
* Vehicle Control: Peptide with buffer and vehicle but no test chemical
* Test Chemical Controls: Test chemical in vehicle and buffer without peptide
* Blank Control: Vehicle and buffer without test chemical or peptide
82
What is required for laboratories before routine use of the kDPRA?
Laboratories must demonstrate proficiency by testing nine reference substances.
83
What does the OECD/OCDE 442D guideline focus on?
It evaluates skin sensitization through keratinocyte activation using in vitro test methods.
84
What are the validated test methods for skin sensitization under OECD/OCDE 442D?
* KeratinoSens™ (ARENrf2 luciferase assay)
* LuSens (ARENrf2 luciferase assay)
* EpiSensA (gene expression-based assay)
85
What is the test principle of the KeratinoSens™ method?
It evaluates skin sensitization potential by assessing Nrf2-mediated activation of ARE-dependent genes in keratinocytes.
86
What are the key parameters calculated in the KeratinoSens™ test?
* Imax: Maximum average fold induction of luciferase activity
* EC1.5: Concentration where luciferase induction exceeds 1.5-fold
* IC50 & IC30: Concentrations causing 50% and 30% reduction in cell viability
87
What is the significance of a positive control in the KeratinoSens™ test?
The positive control must show a statistically significant induction ≥1.5-fold in at least one concentration (4-64 µM).
88
What kind of chemicals can the KeratinoSens™ test evaluate?
It is suitable for a range of chemicals, including those soluble or forming stable dispersions in the test medium.
89
What should be checked if a biphasic response is observed in the KeratinoSens™ test?
Graphs should be verified for clear dose-response curves.
90
Fill in the blank: The kDPRA requires a test chemical to be soluble at _______.
20 mM
91
True or False: The KeratinoSens™ test can determine sensitizer potency.
False
92
What must be done if the fluorescence measurement shows significant interference?
High autofluorescence may require checking for time-dependent depletion to assess its effect.
93
What is the acceptance criterion for vehicle control variance in the kDPRA?
Variance should be <12.5% for at least 5 of the 6 exposure times.
94
What is the acceptance criterion for the positive control in the KeratinoSens™ test?
Positive control (cinnamic aldehyde) must show a statistically significant induction ≥1.5-fold in at least one concentration (464 µM)
## Footnote
EC1.5 of positive control should be within historical mean ±2 SD
95
What are the requirements for a positive KeratinoSens™ result?
A positive result requires at least two of three repetitions meeting:
* Imax ≥1.5-fold with statistical significance
* Viability >70% at EC1.5 concentration
* EC1.5 <1000 µM (or <200 µg/mL for undefined MW chemicals)
* Dose-dependent increase in luciferase induction (or verified biphasic response)
## Footnote
Imax refers to the maximum induction observed
96
What indicates inconclusive results in the KeratinoSens™ test?
Inconclusive results occur if:
* A clear dose-response is missing
* Maximum concentration is <1000 µM and viability remains >70%
* Test chemical induces luciferase activity near cytotoxic levels
## Footnote
Additional testing may be needed for clarification
97
What is the principle of the LuSens test method?
The LuSens test method uses an immortalized human keratinocyte cell line with a luciferase reporter gene controlled by the antioxidant response element (ARE) of the rat NQO1 gene
## Footnote
The luciferase signal indicates the activation of Nrf2-dependent genes by skin sensitisers
98
What are the key steps in the LuSens testing procedure?
The procedure includes:
* Cell Preparation
* Test Chemical and Controls
* Testing Procedure
* Cytotoxicity Assay
* Data Interpretation
## Footnote
Each step ensures accurate assessment of skin sensitization potential
99
What is the overall accuracy of the EpiSensA method when compared to the LLNA?
EpiSensA has an overall accuracy of 83.3% when compared to the LLNA
## Footnote
EpiSensA is validated through independent peer review
100
What are the limitations of the EpiSensA method?
Limitations include:
* Some surfactants can cause false positives
* Chemicals affecting RNA isolation or cell viability below 80% may not be suitable
* Results should be interpreted cautiously under high osmotic stress
## Footnote
High concentrations may induce nonspecific gene expression
101
What does the hCLAT method measure?
The hCLAT method measures the expression of CD86 and CD54 markers in THP1 cells to differentiate between skin sensitizers and nonsensitizers
## Footnote
This indicates monocyte activation, mimicking dendritic cell activation
102
What is the sensitivity and specificity of the hCLAT method?
Sensitivity: 93%, Specificity: 66% compared to LLNA
## Footnote
More likely to produce false negatives for low-to-moderate sensitizers (UN GHS 1B)
103
What is required for a test chemical to be considered positive in the EpiSensA method?
At least one marker gene must exceed specific cutoff values (e.g., ATF3, 1.5-fold) with adequate cell viability
## Footnote
Cell viability must be ≥80%
104
What is the definition of a skin sensitizer according to the UN GHS?
A skin sensitizer is a substance that causes an allergic skin reaction
## Footnote
This is part of the biological events outlined in the Adverse Outcome Pathway (AOP)
105
What are the methods included in the in vitro assays to assess activation of dendritic cells?
Included methods:
* Human Cell Line Activation Test (hCLAT)
* U937 Cell Line Activation Test (USENS™)
* Interleukin8 Reporter Gene Assay (IL8 Luc assay)
* Genomic Allergen Rapid Detection (GARD™skin)
## Footnote
These methods help differentiate between skin sensitisers and nonsensitisers
106
What is a key limitation of DC activation tests in skin sensitization?
DC activation tests represent only one key event in the AOP and may not fully determine a chemical's skin sensitization potential
## Footnote
They should be combined with other data for comprehensive assessment
107
What is required for laboratories before routine use of the LuSens method?
Laboratories must demonstrate proficiency with ten Proficiency Substances
## Footnote
This ensures reliability in testing results
108
What does hCLAT stand for?
Human Cell Line Activation Test
## Footnote
hCLAT is an in vitro assay measuring CD86 and CD54 marker expression.
109
What is the main purpose of the hCLAT test?
To assess monocyte activation by measuring CD86 and CD54 expression on THP1 cells after exposure to a test chemical.
110
What cell line is used in the hCLAT method?
THP1 cells (human monocytic leukemia cell line).
111
What are the two main markers measured in the hCLAT test?
* CD86
* CD54
112
What is the significance of CD86 and CD54 expression in the hCLAT test?
They indicate monocyte activation, mimicking dendritic cell activation crucial for T-cell priming.
113
What technique is used to detect marker changes in the hCLAT assay?
Flow cytometry using fluorochrome-tagged antibodies.
114
What is assessed to ensure marker upregulation occurs at nontoxic concentrations in hCLAT?
Cytotoxicity.
115
What must laboratories demonstrate before routinely using the hCLAT test?
Proficiency with 10 Proficiency Substances.
116
What is the CV75 value in the context of the hCLAT method?
The concentration of test chemical that results in 75% cell viability.
117
What are the incubation conditions for the hCLAT test?
37°C, 5% CO₂ for 24 hours.
118
What is the maximum propagation time for THP1 cells in culture?
2 months (≤ 30 passages).
119
What is the purpose of the U-SENS™ method?
To detect CD86 expression in U937 cells to differentiate skin sensitisers from non-sensitisers.
120
What is the accuracy of the U-SENS™ method compared to LLNA?
86% accuracy (91% sensitivity, 65% specificity).
121
What is the recommended incubation time for the U-SENS™ method?
45±3 hours.
122
What is the main limitation of the U-SENS™ method?
Surfactants may cause false positives.
123
What does the IL-8 Luc assay measure?
IL-8 expression to distinguish skin sensitisers from non-sensitisers.
124
What is the accuracy of the IL-8 Luc assay for skin sensitiser identification?
83.6% accuracy.
125
What is a key limitation of the IL-8 Luc assay?
False negatives are more common for low-potency sensitizers.
126
What type of cells does the GARD™skin method utilize?
SenzaCell™ line, a subclone of the MUTZ-3 cell line.
127
What is the primary goal of the GARD™skin method?
To identify skin sensitisers by analyzing gene expression patterns.
128
What is a critical aspect of the hCLAT procedure?
Cells must pass a reactivity check before testing.
129
Fill in the blank: The hCLAT test classifies chemicals as _______ or nonsensitisers.
sensitisers
130
What does the GARD™skin method analyze to identify skin sensitisers?
Gene expression patterns in the SenzaCell™ line
## Footnote
The SenzaCell™ line is a subclone of the MUTZ-3 cell line.
131
How many genes are involved in the GARDskin Genomic Prediction Signature (GPS)?
196 genes
132
What is the accuracy of the GARDskin method in validation studies?
91.7% accuracy
## Footnote
The accuracy improves to 95.4% when using truly naïve chemicals.
133
What is the sensitivity of the GARDskin method?
92.4%
134
What is the specificity of the GARDskin method?
90.1%
135
What are the ranges for within-laboratory reproducibility (WLR) for the GARDskin method?
78.6% to 89.2%
136
What are the ranges for between-laboratory reproducibility (BLR) for the GARDskin method?
82.1% to 92.0%
137
What type of substances is the GARDskin method suitable for testing?
Mono-constituent substances
## Footnote
It is also technically applicable to multi-constituent substances and mixtures.
138
What are the known limitations of the GARDskin method?
Issues with solubility, vehicle compatibility, and potential interference from autofluorescent chemicals.
139
What type of algorithm does the GDAA use to analyze data in the GARDskin method?
Support Vector Machine (SVM) algorithm
140
What is the primary function of the GARDskin method?
Identifying skin sensitisation hazards
141
What is the recommended framework for using the GARDskin method?
Integrated Approaches to Testing and Assessment (IATA)
142
Fill in the blank: The GARDskin method uses the _______ cell line as an in vitro model for dendritic cells.
SenzaCell™
143
True or False: The GARDskin method can only be used for testing single chemicals.
False
144
What is measured from isolated RNA in the GARDskin method?
Gene expression levels of the GARDskin GPS
145
What is the duration of the exposure of cells to test chemicals in the GARDskin method?
24 hours
146
What type of chemicals does the GARDskin method work well with?
Various chemical types, including lipophilic compounds, indirect haptens, and metal compounds.
