OECD Questions Flashcards
(139 cards)
1
Q
Q1. In an OECD TG 408 90-day oral toxicity study, what is the minimum number of animals per dose group recommended for statistical validity?
A. 5 animals (males only)
B. 10 animals (5 male, 5 female)
C. 20 animals (10 male, 10 female)
D. 40 animals (20 male, 20 female)
A
Correct Answer: C (At least 20 animals, 10 of each sex, per group)
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Q2. OECD 408 studies typically include at least three dose levels plus a control. What is the purpose of the highest dose level in a 90-day study?
A. To cause mortality in the majority of animals
B. To induce some toxic effects without severe suffering or death
C. To produce no observable effect
D. To equal the median lethal dose (LD₅₀)
A
Correct Answer: B (The top dose is chosen to induce some maternal or systemic toxicity but not death or severe suffering)
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Q3. What is the typical “limit dose” in an OECD 408 subchronic (90-day) oral toxicity test, above which a full study might not be needed if no effects are seen?
A. 50 mg/kg/day
B. 300 mg/kg/day
C. 1000 mg/kg/day
D. 5000 mg/kg/day
A
Correct Answer: C (1000 mg/kg body weight per day)
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Q4. Which of the following endpoints are routinely measured in a 90-day OECD 408 study?
A. Body weight and food consumption, clinical observations, hematology, clinical chemistry, organ weights, and histopathology
B. Only mortality and gross necropsy
C. Behavioral cognitive testing and ophthalmic ERG recordings
D. Long-term carcinogenicity occurrence
A
Correct Answer: A (Body weights, food/water intake, daily clinical signs, ophthalmological exam, hematology, biochemistry, urinalysis, followed by necropsy and histopathology)
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Q5. What is the primary goal of the OECD TG 408 90-day study in rodents?
A. To determine the acute LD₅₀ of the substance
B. To identify major toxic effects and target organs, and establish a No-Observed-Adverse-Effect Level (NOAEL)
C. To evaluate carcinogenic potential over a lifetime
D. To assess reproductive performance across generations
A
Correct Answer: B (It provides information on major toxic effects, target organs, and helps establish a subchronic NOAEL)
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Q1. What is the main purpose of the OECD TG 414 prenatal developmental toxicity study?
A. To evaluate chemical effects on adult male fertility only
B. To assess effects of prenatal exposure on the pregnant animal and developing fetus, including teratogenicity and embryo-fetal lethality
C. To determine carcinogenic effects in offspring after birth
D. To measure acute maternal toxicity in non-pregnant females
A
Correct Answer: B (It provides information on effects of prenatal exposure on the pregnant test animal and on embryo-fetal development, identifying developmental toxicity and maternal toxicity)
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Q2. In OECD 414, how many pregnant females per group are typically required to ensure valid results?
A. ~20 pregnant females per dose (with at least 16 with implantation sites at term)
B. 6 pregnant females per group
C. Only one litter (from one female) per dose level
D. No females; it’s an in vitro test
A
Correct Answer: A (Each dose and control group should have enough mated females to yield about 20 pregnant females with implantations; groups with fewer than 16 pregnant females may be insufficient)
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Q3. When are test substances administered to animals in an OECD 414 study?
A. During the entire lactation period only
B. Daily from implantation through the day before scheduled caesarean section (covering the major period of organogenesis)
C. Only on the day of mating
D. From birth of the F1 generation until weaning
A
Correct Answer: B (Dosing is typically daily from implantation (e.g. gestation day 5 post-mating) to the day prior to scheduled caesarean section, i.e., through organogenesis up to just before parturition)
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Q4. Which endpoints are evaluated in the fetuses in an OECD TG 414 study?
A. Fetal weight, sex, external malformations, visceral abnormalities, skeletal defects, and counts of implantations, resorptions, and live/dead fetuses
B. Adult behavior and cognitive function of offspring
C. Carcinogenic lesions in adult offspring
D. Only the number of live pups, without examining malformations
A
Correct Answer: A (Typical fetal endpoints include number of implantations, early/late resorptions, live and dead fetuses, fetal weight and sex, and examination for external, visceral, and skeletal anomalies)
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Q5. What defines a “developmental toxicant” outcome in OECD 414?
A. Any maternal death during gestation
B. Findings of either embryo-fetal death, growth retardation, or structural malformations caused by the test substance (at doses that also may cause maternal toxicity)
C. Only malformations that occur in the absence of any maternal toxicity
D. Postnatal behavioral deficits in offspring
A
Correct Answer: B (A positive developmental toxicity is indicated by adverse effects on the embryo or fetus – such as death, altered growth, or malformations – which may occur with or without maternal toxicity. Teratogenic effects are structural malformations in the fetus.)
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Q6. True or False: OECD 414 allows a limit test at a high dose (e.g. 1000 mg/kg/day) if no toxicity is expected, potentially negating the need for a full multiple-dose study.
A
Correct Answer: True. (If a single dose at at least 1000 mg/kg/day causes no observable maternal or developmental toxicity, a full study with three doses may not be necessary) .
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Q1. The OECD TG 420 Fixed Dose Procedure for acute oral toxicity is characterized by which of the following?
A. Testing both sexes in large groups to find an exact LD₅₀
B. Testing primarily one sex (usually female rats) in a stepwise manner at fixed dose levels, aiming to observe clear toxicity signs rather than lethality
C. Dosing a single animal at increasing doses until death occurs
D. Using cell cultures to estimate oral toxicity
A
Correct Answer: B (TG 420 uses one sex – usually females – and a sequential dosing at fixed levels (5, 50, 300, 2000 mg/kg, etc.) to identify the dose causing clear signs of toxicity without lethality, thereby avoiding death as an endpoint) .
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Q2. What are the standard fixed dose levels used in the OECD 420 acute oral toxicity test (main study)?
A. 50, 500, 5000, 15000 mg/kg
B. 5, 50, 300, 2000 mg/kg (with 5000 mg/kg as an optional limit)
C. 1, 10, 100, 1000 mg/kg
D. Any doses chosen by the investigator
A
Correct Answer: B (The fixed dose procedure uses 5, 50, 300, and 2000 mg/kg as the default dose levels, with 5000 mg/kg sometimes used as an additional limit dose if required) .
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Q3. In the Fixed Dose Method (OECD 420), what criterion is used to classify a substance’s acute toxicity hazard?
A. The exact LD₅₀ value is calculated from mortality data
B. The presence or absence of gross necropsy findings
C. The occurrence of clear clinical signs of toxicity at one of the fixed dose levels (which corresponds to a GHS category)
D. The substance’s color change in the stomach
A
Correct Answer: C (Classification is based on whether animals show evident but non-lethal toxicity at specific fixed doses. The absence of serious toxicity up to a certain dose, or the presence of toxicity at a particular dose, places the substance into a Globally Harmonized System acute toxicity category without needing an exact LD₅₀) .
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Q4. Which statement about animal welfare is TRUE for OECD TG 420?
A. It requires at least 20 animals per dose.
B. It aims to avoid animal death as an endpoint by stopping at doses that cause clear signs (humane endpoints are used for moribund animals) .
C. It does not use any humane endpoints.
D. It uses more animals than the older OECD 401 method.
A
Correct Answer: B (The fixed dose procedure was developed to reduce animal suffering by avoiding lethal outcomes. Doses expected to cause severe pain or death are avoided; if an animal shows severe distress, it is humanely euthanized and considered as if it died for the purpose of results. Overall, OECD 420 uses fewer animals than the old LD₅₀ test) .
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Q5. True or False: Under OECD 420, if no toxicity is observed in an initial sighting study at 2000 mg/kg, the test chemical can be classified as having low acute toxicity (e.g., GHS “Unclassified” above Category 4).
A
Correct Answer: True. (If a single animal (sighting) and confirmatory group show no significant toxicity at 2000 mg/kg, the substance is considered relatively non-toxic acutely, often not classified in Categories 1–4 of acute toxicity).
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Q1. What is the primary purpose of the in vitro 3T3 Neutral Red Uptake (NRU) phototoxicity test (OECD TG 432)?
A. To identify substances that cause genetic mutations when exposed to light
B. To identify the phototoxic potential of chemicals – i.e. toxicity elicited or enhanced after exposure to light (typically UVA)
C. To measure skin irritation in vitro
D. To detect acute oral toxicity under UV exposure
A
Correct Answer: B (The 3T3 NRU phototoxicity assay detects compounds that are non-toxic in the dark but become toxic to cells upon light exposure. It measures phototoxic potential as reduced cell viability with UVA vs without) .
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Q2. Which cell type is used in the OECD TG 432 phototoxicity test?
A. Human keratinocytes in a 3D model
B. Bovine corneal cells
C. Mouse Balb/c 3T3 fibroblast monolayers
D. Bacterial cells (E. coli)
A
Correct Answer: C (The test uses a mouse Balb/c 3T3 fibroblast cell line, grown as a monolayer, to assess cytotoxicity with and without UVA light) .
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Q
Q3. How is a positive phototoxic response identified in the 3T3 NRU assay?
A. By a high Photo-Irritation Factor (PIF) or Mean Photo Effect (MPE), indicating much greater cytotoxicity with light than without .
B. If cells become fluorescent under UV
C. If the test substance changes color upon irradiation
D. By any cytotoxicity in either dark or light conditions
A
Correct Answer: A (Data are analyzed by comparing IC₅₀ values ± UVA. A substance with PIF > 5 or MPE > 0.15 is predicted as phototoxic, while lower ratios indicate “no phototoxicity” or “probable” phototoxicity in between) .
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Q4. True or False: The 3T3 NRU phototoxicity test includes an exogenous metabolic activation system (like S9) to simulate metabolism.
A
Correct Answer: False. (Metabolic activation is generally not included in this phototoxicity test because most known phototoxins do not require metabolic activation to exert phototoxic effects, and adding such systems isn’t considered necessary) .
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Q5. Which of the following is NOT addressed by the OECD 432 phototoxicity assay?
A. Acute phototoxic effects (photo-cytotoxicity)
B. Photoallergic reactions of the immune system
C. Identification of UV-absorbing chemicals that might cause skin phototoxicity
D. Phototoxic hazard screening for cosmetics and chemicals
A
Correct Answer: B (The 3T3 NRU test is designed for acute phototoxicity. It does not detect photoallergy or photocarcinogenesis, which involve immune-mediated or long-term effects not measured by this assay) .
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Q
OECD TG 437 – Bovine Corneal Opacity and Permeability (BCOP) Test
Q1. What is the primary use of the OECD TG 437 BCOP test?
A. To fully replace all eye irritation testing including mild irritants
B. To identify chemicals that cause serious eye damage (corrosives/severe irritants, UN GHS Category 1) without using live animals
C. To determine skin corrosion potential
D. To measure photochemical damage in eyes
A
Correct Answer: B (The BCOP is an in vitro/ex vivo assay using bovine corneas, intended mainly to classify substances as ocular corrosives or severe irritants (Category 1) so that they can be labeled appropriately without animal testing) .
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Q
Q2. In the BCOP assay, how is ocular damage quantified?
A. By counting dead corneal cells under a microscope
B. By measuring corneal opacity (light transmission) and permeability to fluorescein dye, combined into an In Vitro Irritancy Score (IVIS)
C. By observing the cornea for color change
D. By scoring tear production in eyes
A
Correct Answer: B (Corneal opacity is measured instrumentally (reduced light transmission), and permeability is measured by sodium fluorescein dye penetration. These are combined to calculate an IVIS for each test substance) .
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Q3. Which outcome in the BCOP test would lead to a classification as “serious eye damage” (GHS Category 1)?
A. IVIS (In Vitro Irritancy Score) significantly above 55
B. IVIS around 10
C. Any visible change to the cornea, regardless of score
D. A negative IVIS (below 0)
A
Correct Answer: A (An IVIS > 55 indicates severe irritancy/corrosivity, warranting Category 1 classification under UN GHS) . (By contrast, an IVIS ≤ 3 suggests no need for classification (No Category) .)
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Q4. True or False: A chemical that yields an IVIS of 2 in the BCOP test can be confidently labeled as “non-irritant to eyes” without further testing.
Correct Answer: True. (In OECD 437, substances with IVIS ≤ 3 are considered “No Category” (non-irritant) for eye irritation . However, regulatory use of a negative result may depend on the testing strategy. BCOP is good at identifying non-irritants and severe irritants, but less reliable in distinguishing mild/moderate irritants .)
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Q5. What is a limitation of the BCOP assay regarding classification of eye irritants?
A. It cannot detect any severe irritants
B. It tends to over-predict mild irritants as more severe and under-predict some chemicals that cause reversible irritation
C. It is too expensive to perform
D. It uses live animals, raising ethical issues
Correct Answer: B (The BCOP may not be adequately validated for mild or moderate irritants – it can overestimate their hazard or miss some specific types. Thus a negative (non-corrosive) result often triggers additional testing for classification into Category 2 or no-category) .
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Q6. In the BCOP test protocol, from where are the corneas obtained and how does this align with 3Rs principles?
A. From live rabbits, which contradicts 3Rs
B. From slaughtered cattle eyes (abattoir by-products), aligning with Reduction and Replacement (no additional animals euthanized solely for the test)
C. From human donors
D. The test does not use real corneas
Correct Answer: B (Bovine eyes from slaughterhouses are used, which implements the Replacement of live animal testing and makes use of tissue that would otherwise be waste, in line with 3Rs principles) .
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OECD TG 438 – Isolated Chicken Eye (ICE) Test Method
Q1. What is the purpose of the OECD TG 438 Isolated Chicken Eye test?
A. To identify chemicals causing serious eye damage (corrosives/severe irritants) without using live animals
B. To test skin sensitization in chickens
C. To measure avian systemic toxicity
D. To replace BCOP because it’s less variable
Correct Answer: A (Like BCOP, the ICE method is used primarily for identifying ocular corrosives and severe irritants (Category 1), using enucleated chicken eyes as an ex vivo model) .
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Q2. Which measurements are taken in the ICE test to evaluate eye irritation?
A. EEG recordings from chicken brains
B. Corneal opacity (degree of cloudiness), corneal swelling (thickness increase), fluorescein retention (permeability), and observation of corneal surface damage
C. Tear fluid pH changes
D. Number of dead corneal cells only
Correct Answer: B (The ICE protocol assesses corneal opacity qualitatively, corneal swelling quantitatively (percentage thickness increase), fluorescein dye retention, and any macroscopic damage to the cornea. These endpoints are then combined to derive an irritation classification) .
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Q3. In the ICE test, what combination of findings would typically indicate a test substance is a “serious eye irritant/corrosive” (Category 1)?
A. Minor opacity and no fluorescein staining
B. Severe corneal opacity (score 4) in at least one eye or opacity ≥3 in two eyes, and/or severe epithelial damage (sloughing)
C. Only transient redness in conjunctiva
D. Any positive result triggers Category 1
Correct Answer: B (Category 1 classification in ICE is triggered by severe effects such as corneal opacity score of 4 in at least one eye or high opacity (≥3) observed early, significant fluorescein retention (score 3), or severe loosening of epithelium. The ICE has defined combinations of these endpoint scores that result in a prediction of corrosive/severe irritant) .
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Q4. True or False: The ICE test is considered adequately validated for identifying mild or moderate eye irritants (GHS Category 2).
Correct Answer: False. (OECD 438 is primarily accepted for identifying Category 1 severe irritants. It is not currently considered sufficiently validated to distinguish mild/moderate irritants from non-irritants . A negative result in ICE does not automatically mean a chemical is “non-irritant” without further testing.)
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Q5. From what source are the eyes used in the OECD 438 ICE assay obtained?
A. Live chickens specifically sacrificed for the test
B. Abattoir (slaughterhouse) chickens, typically ~7 weeks old, as a by-product of poultry production
C. Donated human eyes
D. Synthetic polymer eye models only
Correct Answer: B (Chicken eyes used in the ICE assay are collected from poultry slaughterhouses (approximately 7-week-old chickens). This uses waste material and avoids sacrificing animals solely for testing, in accordance with 3Rs principles) .
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OECD TG 442A – Skin Sensitization: Local Lymph Node Assay (LLNA) – DA
Q1. OECD 442A describes the LLNA: DA (Local Lymph Node Assay: DA). What does “DA” refer to in this context?
A. “Dose Adjustment” version of LLNA
B. “Dominant Allergen” method
C. Daicel ATP assay – a non-radioactive luminescence adaptation of the LLNA
D. “Delayed Absorption” modification
Correct Answer: C (442A is the LLNA: DA, which stands for the Daicel Adenosine triphosphate assay or “Draining auricular lymph node – ATP” assay. It measures lymphocyte proliferation by a bioluminescent method (ATP content) instead of radioactive incorporation) .
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Q2. The LLNA: DA (OECD 442A) and LLNA: BrdU (OECD 442B) share what advantage over the traditional LLNA (OECD 429)?
A. They use guinea pigs instead of mice
B. They avoid the use of radioactive materials (no ^3H-thymidine) by using alternative endpoints for lymphocyte proliferation
C. They do not require euthanasia of animals
D. They can be done entirely in vitro
Correct Answer: B (Both 442A and 442B are non-radioactive modifications of the mouse LLNA. 442A uses a luminescence (ATP) endpoint, and 442B uses bromodeoxyuridine (BrdU) incorporation with ELISA or flow cytometry, thereby eliminating the need for radioactive tracers) .
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Q3. In the context of skin sensitization testing, what does the Local Lymph Node Assay measure as an indicator of sensitization?
A. Irritant skin damage
B. Lymphocyte proliferation in lymph nodes draining the chemical application site (indicative of an immune response)
C. Antibody production in blood serum
D. Pain response in mice
Correct Answer: B (The LLNA measures proliferation of lymphocytes in the auricular (ear-draining) lymph nodes after topical application of a test substance to the ears. A significant proliferation (stimulation index ≥ threshold) indicates the substance has skin sensitizing potential.)
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Q4. True or False: OECD 442A still involves live animals.
Correct Answer: True. (LLNA: DA is an in vivo test in mice – it refines the traditional LLNA to reduce hazard (no radioisotopes) and possibly reduce animal numbers, but it still uses mice to assess immune response.)
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Q1. What is the key feature of the OECD 442B LLNA BrdU-ELISA method?
A. It uses guinea pigs and measures skin redness
B. It quantifies lymph node cell proliferation by incorporating 5-bromo-2′-deoxyuridine (BrdU) into DNA and detecting it via ELISA or flow cytometry
C. It measures ATP content in lymph nodes
D. It is an in vitro assay using human cells
Correct Answer: B (The 442B method is a non-radioactive LLNA where proliferating lymphocytes incorporate BrdU. The BrdU is then detected with an ELISA or via a fluorescence-tagged antibody in flow cytometry, indicating the level of cell proliferation in response to the test chemical) .
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Q2. Compared to the guinea pig maximization test (OECD 406), what is an advantage of the LLNA (including OECD 442A/442B variants)?
A. LLNA can provide a quantitative dose–response (stimulation index) and is generally less time-consuming and uses fewer animals than guinea pig tests .
B. LLNA requires induction and challenge phases over several weeks
C. LLNA detects irritants better than sensitizers
D. There is no advantage; guinea pig tests are superior
Correct Answer: A (The mouse LLNA and its variants reduce animal use and time, allow quantitative estimation of sensitizing potency (via stimulation index values), and avoid the pain of adjuvant injections used in guinea pig tests . These methods are also in line with 3Rs, refining the procedure by using fewer animals and causing less distress.)
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Q3. In an LLNA test (OECD 442B), a Stimulation Index (SI) is calculated. What SI value is generally used as a cutoff to consider a substance a sensitizer in the traditional LLNA (OECD 429)?
A. SI ≥ 3
B. SI ≥ 1
C. Any SI > 0
D. SI < 1.6
Correct Answer: A (Traditionally, an SI of 3 or more (meaning a three-fold or greater proliferation in treated groups compared to vehicle control) is the threshold for a positive LLNA, indicating skin sensitization potential. Note: Some protocols or recent evaluations suggest SI ≥1.6 for certain non-radioactive LLNA variants , but ≥3 is the classic criterion for OECD 429.)
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OECD TG 442C – Skin Sensitization: In Chemico Direct Peptide Reactivity Assay (DPRA)
Q1. What key event in the skin sensitization adverse outcome pathway is addressed by the Direct Peptide Reactivity Assay (OECD TG 442C)?
A. Keratinocyte inflammation (KE2)
B. Covalent binding of the chemical to skin proteins (haptenation – the molecular initiating event, KE1)
C. Dendritic cell migration (KE4)
D. T-cell proliferation (KE4)
Correct Answer: B (The DPRA is an in chemico assay that models the molecular initiating event of skin sensitization: the covalent binding of electrophilic substances to nucleophilic amino acid residues in proteins) . It measures the reactivity of a test chemical with cysteine and lysine peptides.
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Q2. How is the outcome of the DPRA measured?
A. By determining the amount of peptide remaining or depleted after reaction with the test substance, typically via HPLC analysis
B. By observing mouse lymph node swelling
C. By measuring ATP in a cell line
D. By assessing skin redness on a human patch test
Correct Answer: A (In DPRA, synthetic peptides containing cysteine or lysine are exposed to the test chemical. The percentage of peptide depletion is quantified (often by HPLC/UV). High depletion of the peptide indicates the chemical is reactive and thus potentially a skin sensitizer) .
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Q3. What result in a DPRA would be interpreted as a positive sensitizer prediction?
A. No peptide depletion (<~5%)
B. Significant peptide depletion (e.g. high reactivity classification such as >42% cysteine/lysine peptide depletion)
C. The test chemical precipitates in the assay
D. The assay is not relevant for sensitization
Correct Answer: B (A chemical that substantially depletes the peptides is considered reactive. For example, OECD DPRA has thresholds: chemicals are categorized as having high, moderate, low or no peptide reactivity. High reactivity (e.g. >42% peptide depletion) or moderate reactivity above certain cut-offs is predictive of skin sensitization potential.)
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Q4. True or False: The DPRA (OECD 442C) is an in chemico method that does not use live cells or animals.
Correct Answer: True. (DPRA is performed entirely in solution with synthetic peptides, so it involves no live cells or animals – making it an in chemico test addressing the first step of the sensitization pathway) .
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Q5. Which two amino acid residues are the synthetic peptides in the standard DPRA designed to represent?
A. Histidine and arginine
B. Cysteine and lysine (nucleophilic sites common in skin proteins)
C. Glycine and alanine
D. Serine and threonine
Correct Answer: B (The DPRA uses a cysteine-containing peptide and a lysine-containing peptide as proxies for proteins in the skin. Many sensitizing chemicals are electrophiles that react with cysteine (thiol) or lysine (amine) side chains) .
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OECD TG 442D – Skin Sensitization: In Vitro ARE-Nrf2 Luciferase Test (KeratinoSens)
Q1. What biological process is the KeratinoSens™ assay (OECD 442D) designed to detect as part of the skin sensitization pathway?
A. Protein binding in a chemical tube
B. Activation of epidermal keratinocytes (specifically the oxidative stress response/ARE-Nrf2 pathway, Key Event 2)
C. T-cell proliferation in lymph nodes
D. Histamine release from mast cells
Correct Answer: B (KeratinoSens is a reporter gene assay using an immortalized human keratinocyte cell line with a luciferase gene under control of an Antioxidant Response Element (ARE). It detects activation of the cellular stress response in keratinocytes – indicative of a sensitizer’s ability to induce cytokines and danger signals, which is the second key event in the skin sensitization AOP) .
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Q2. In the OECD 442D KeratinoSens assay, a positive result is typically defined by:
A. A significant induction of luciferase activity (above a threshold, e.g. ≥1.5-fold) at sub-cytotoxic concentrations, in a dose-responsive manner
B. Observation of cell death under a microscope
C. Lymph node activation
D. Peptide depletion measurement
Correct Answer: A (KeratinoSens yields a positive prediction if the test chemical induces the luciferase reporter above a certain fold-increase (e.g. 1.5 or 2 fold over baseline, depending on protocol) at concentrations that do not severely reduce cell viability (viability should remain >70%). Typically, reproducible induction at multiple concentrations is required) .
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Q3. The KeratinoSens test system uses which cell line?
A. Mouse lymphoma cells
B. Human keratinocyte-derived cell line (HaCaT or similar) engineered with a luciferase reporter for ARE-driven genes
C. Human T-cells
D. Mouse spleen cells
Correct Answer: B (It uses a human keratinocyte cell line with an antioxidant response element (ARE)-linked luciferase reporter. Upon exposure to sensitizers that activate the Nrf2-ARE pathway, the cells produce luciferase, which is measured as an indicator of KE2 – keratinocyte activation) .
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Q4. True or False: OECD 442D (KeratinoSens) can by itself distinguish between strong and weak sensitizers in terms of potency categories.
Correct Answer: False. (While KeratinoSens can give some indication (e.g. by the concentration needed to reach a certain induction), it is not a direct measure of in vivo potency. It is used qualitatively (sensitizer vs non-sensitizer). For quantitative potency categorization, an integrated approach or other assays would be needed. OECD 442D is generally used as a yes/no screen as part of an integrated testing strategy.)
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Q5. What key regulatory application does OECD 442D (and related assays 442C/442E) serve?
A. Stand-alone approval of new drugs
B. Replacement of animal test (LLNA) for skin sensitization hazard classification and risk assessment, as part of defined approaches or IATA (Integrated Approaches to Testing and Assessment) for sensitization
C. Determining phototoxicity of chemicals
D. Assessing acute oral toxicity
Correct Answer: B (These in vitro/in chemico assays are used to replace or minimize animal use for skin sensitization. OECD 442C, 442D, 442E are often used in combination under Defined Approaches (e.g. OECD TG 497 for skin DA) or IATA to hazard classify chemicals as skin sensitizers or not, in lieu of LLNA) .
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OECD TG 442E – Skin Sensitization: Human Cell Line Activation Test (h-CLAT)
Q1. The h-CLAT (OECD 442E) assay is intended to model which key event in the skin sensitization pathway?
A. Keratinocyte cytokine release
B. Dendritic cell activation (Key Event 3) – specifically upregulation of costimulatory surface markers on dendritic cells
C. T-cell proliferation
D. IgE antibody production
Correct Answer: B (The Human Cell Line Activation Test targets the activation of dendritic cells, the third key event. It measures changes in expression of cell surface molecules (CD86 and CD54) on a human monocytic cell line (THP-1), which serves as a surrogate for dendritic cells) .
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Q2. In the OECD 442E h-CLAT, what indicates a positive result suggestive of a skin sensitizer?
A. A significant increase in expression of CD86 and/or CD54 on THP-1 cells relative to control (above defined thresholds, e.g. RFI ≥ 150% for CD86 or ≥ 200% for CD54)
B. Cell death of >90%
C. Secretion of histamine by THP-1 cells
D. Depletion of peptides in solution
Correct Answer: A (The h-CLAT defines positivity by upregulation of the co-stimulatory molecules: typically, if the Relative Fluorescence Intensity (RFI) of CD86 ≥150% or CD54 ≥200% at any tested non-cytotoxic concentration in two independent runs, the chemical is considered a sensitizer. Increased expression of these markers is indicative of dendritic cell activation) .
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Q3. What type of cells are used in the h-CLAT assay?
A. Primary human skin dendritic cells from donors
B. A human monocytic leukemia cell line (THP-1) that can be stimulated to mimic dendritic cell surface marker expression
C. Mouse lymph node cells
D. Keratinocytes with a luciferase gene
Correct Answer: B (h-CLAT uses the THP-1 human monocytic cell line, which upon exposure to sensitizers will upregulate surface markers CD54 and CD86, mimicking the phenotype of activated dendritic cells) .
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Q4. The combination of assays DPRA (442C) + KeratinoSens (442D) + h-CLAT (442E) in an Integrated Testing Strategy is often sufficient to:
A. Fully replace the need for any in vivo skin sensitization test (LLNA) for many substances
B. Identify photoallergens
C. Classify eye irritation
D. Determine skin corrosion potential
Correct Answer: A (Using multiple non-animal assays that address different key events of the sensitization pathway (chemistry-based haptenation, keratinocyte activation, dendritic cell activation) can provide an accurate prediction of skin sensitization. Defined approaches using some or all of these tests (OECD TG 497 on skin sensitization defined approaches) are accepted as full replacements for LLNA in many regulatory contexts) .
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Q5. True or False: OECD TG 442E h-CLAT is an in vitro assay that does not involve any live animals.
Correct Answer: True. (h-CLAT is entirely in vitro, using a human cell line. It aligns with Replacement in the 3Rs by avoiding animal use, and is part of the toolbox to replace animal tests for skin sensitization) .
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OECD TG 443 – Extended One-Generation Reproductive Toxicity Study (EOGRTS)
Q1. How does the OECD TG 443 Extended One-Generation Reproductive Toxicity Study differ from the traditional two-generation reproduction study (OECD 416)?
A. It examines only one sex of animal
B. It includes only one generation of offspring (F1), with no routine breeding of a second generation, unless triggered, thereby using fewer animals .
C. It does not assess fertility endpoints
D. It is shorter and ends at mating, not including pregnancy or lactation
Correct Answer: B (The EOGRTS involves one generation of offspring. F1 animals are observed and assessed through development, and a second generation (F2) is not automatically produced – it would only be bred if specific triggers (e.g. effects on reproduction or development) indicate a need. This design reduces animal use compared to a two-generation study while expanding data collection on the first generation) .
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Q2. Which additional evaluations can OECD 443 incorporate that are not typically covered in a standard two-generation study?
A. Developmental neurotoxicity and immunotoxicity assessments in subsets of the F1 generation (via optional Cohort 2 for neurodevelopment, and Cohort 3 for immune function)
B. Only general toxicity in parents
C. Carcinogenicity in F2 animals
D. Acute toxicity in F1 pups
Correct Answer: A (The “extended” design allows inclusion of specialized cohorts of F1 offspring: e.g., Cohort 2A/2B for developmental neurotoxicity testing (learning, memory, motor activity), and Cohort 3 (with a potential 3A positive control subgroup) for developmental immunotoxicity. These cohorts enable detection of neurodevelopmental or immune system effects within the same study)
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Q3. In an OECD 443 study, at what stages are the F0 (parental) animals exposed to the test chemical?
A. Only during mating
B. During growth/maturation (premating), through mating, throughout gestation, and through lactation until weaning of F1 pups .
C. From conception to birth only
D. Only after the F1 are born
Correct Answer: B (Parent animals (P generation) are dosed for a premating period (to evaluate effects on gametogenesis and cycling), during mating, and (for females) throughout pregnancy and lactation until weaning of the F1. This captures parental fertility, embryonic development, and lactational exposure of offspring) .
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Q4. What reproductive and developmental endpoints are evaluated in the EOGRTS (OECD 443)?
A. Fertility indices (estrous cycles, mating success, conception rates), gestation length, parturition, litter size, survival of pups, growth and development of F1 (including physical development milestones, puberty onset), and potential functional tests (if DNT/DIT cohorts)
B. Only adult toxicity in parents
C. Only teratogenic endpoints at birth
D. Single generation fertility with no offspring examination
Correct Answer: A (OECD 443 is comprehensive: it assesses parental reproductive function (e.g. estrous cyclicity, sperm parameters if collected, mating performance), as well as offspring viability, neonatal health, growth, and developmental milestones. If triggered or included by design, F1 animals can be tested for cognitive function or immunocompetence. Organ weights and histopathology in both P and F1 (at adult age) are done to identify systemic and reproductive organ toxicity) .
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Q5. True or False: The Extended One-Generation study can be used to establish a No-Observed-Adverse-Effect Level (NOAEL) for reproductive and developmental effects, which can inform human risk assessment similarly to the two-generation study.
Correct Answer: True. (EOGRTS yields NOAELs for parental systemic toxicity, reproductive performance, and developmental outcomes in offspring. It is a regulatory replacement for the two-generation study, intended to provide equivalent or better information (including potential endocrine or neuro/immune effects) with fewer animals.)
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Q6. Why is the EOGRTS considered a 3Rs advancement over the traditional two-generation test?
A. It completely eliminates the need for any animal use (False – it still uses animals, but fewer).
B. It reduces animal use by not routinely producing a second generation and can incorporate other toxicological evaluations into one study, thereby avoiding separate studies .
C. It refines by causing more pain to observe effects
D. It only uses in vitro methods
Correct Answer: B (The EOGRTS reduces the total number of animals needed by dropping the second generation unless necessary, and by integrating endpoints like DNT or DIT into one study rather than doing separate studies. It also refines the test design by focusing on the most relevant information and potentially improving welfare compared to conducting multiple separate studies) .
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OECD TG 471 – Bacterial Reverse Mutation Test (Ames Test)
Q1. The OECD TG 471 test is commonly known as the “Ames test.” What is its purpose?
A. To identify substances that cause gene mutations in bacteria (indicating potential genotoxic mutagens)
B. To detect skin sensitizers
C. To measure acute bacterial toxicity
D. To assess eye irritation
Correct Answer: A (The Ames test detects reverse mutations in special strains of bacteria, revealing if a chemical can induce genetic mutations – a screening test for genotoxicity) .
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Q2. How many bacterial strains, and which ones, are recommended in OECD 471 to ensure broad detection of mutagens?
A. One strain of E. coli only
B. At least five strains: four different Salmonella typhimurium strains (TA1535, TA1537 or TA97a, TA98, TA100) plus one E. coli strain (e.g. WP2 uvrA or WP2 uvrA(pKM101))
C. Any two bacteria of the tester’s choice
D. Five strains of Staphylococcus aureus
Correct Answer: B (OECD 471 specifies using a set of Salmonella strains that detect base-pair substitutions and frameshift mutations, and an E. coli strain (or S. typhimurium TA102) to detect certain oxidizing mutagens or cross-linkers. This combination maximizes the mutation types and chemical classes that can be caught) .
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Q3. Why is an S9 metabolic activation system used in many Ames test plates?
A. Bacteria require S9 to grow
B. To simulate mammalian metabolism by providing enzymes that can convert pro-mutagens to mutagens
C. It prevents bacterial contamination
D. It is a source of histidine for the media
Correct Answer: B (The S9 mix, usually liver enzymes from induced rodents, is added to some plates to metabolically activate substances. Some chemicals are not direct mutagens but their metabolites are; the S9 activation mimics metabolism in humans/animals to reveal such mutagens) .
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Q4. In a standard Ames test, a positive result is typically indicated by:
A. Bacterial lawn death
B. A dose-related increase in the number of revertant colonies in treated plates compared to solvent control, in at least one strain ±S9, exceeding a threshold (e.g. at least 2-3 times the spontaneous rate)
C. Color change in the agar
D. Fewer colonies than the control
Correct Answer: B (Mutagenicity is concluded if the test substance causes a reproducible, statistically significant increase in revertant colony counts in one or more tester strains (with or without S9). Typically, at least a doubling of the background mutation rate along with a dose-response is considered positive.)
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Q5. Which of the following is NOT a strain used in the OECD 471 bacterial reverse mutation test?
A. Salmonella typhimurium TA100
B. Salmonella typhimurium TA98
C. Escherichia coli WP2 uvrA
D. Bacillus subtilis 168
Correct Answer: D (Bacillus subtilis is not used in the standard Ames panel. The typical strains are specific Salmonella typhimurium and E. coli strains that have mutations making them sensitive to mutagens by requiring histidine or tryptophan and having DNA repair defects) .
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Q6. True or False: A negative Ames test guarantees that a chemical is not carcinogenic.
Correct Answer: False. (While many carcinogens are mutagenic in the Ames test, some carcinogens act via non-genotoxic mechanisms or are not detected by bacterial systems . Thus, a negative result does not ensure a chemical is non-carcinogenic; further testing (e.g. in mammalian cells or in vivo) may be warranted for a comprehensive assessment.)
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OECD TG 492 – Reconstructed Human Cornea-Like Epithelium (RhCE) Test Method
Q1. What is the main purpose of OECD TG 492 RhCE tests such as EpiOcular™ EIT or SkinEthic™ HCE?
A. To identify chemicals that do not require classification for eye irritation or serious eye damage (i.e., to screen out non-irritants)
B. To identify skin sensitizers
C. To classify Category 1 eye irritants
D. To detect phototoxicants
Correct Answer: A (These human 3D cornea-like tissue assays are primarily used to determine if a substance is a non-irritant (No Category) to the eye, so it can be labeled accordingly or avoid further testing) .
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Q2. In a RhCE eye irritation test (OECD 492), how is irritation potential assessed?
A. By observing the tissue for inflammation
B. By measuring cell viability (e.g. MTT assay) after a defined exposure; low viability indicates the test chemical is irritating to the tissue
C. By looking for color change in the medium
D. By bacterial growth inhibition
Correct Answer: B (RhCE models measure the reduction in cell viability caused by the test chemical. If the mean tissue viability falls below a specified cutoff (e.g. ≤ 60% of the negative control), the substance is considered to have eye irritant potential; if viability remains high (>60%), the substance is predicted as “No Category” (non-irritant)) .
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Q3. Which of the following test systems is not an example of a validated RhCE model for OECD 492?
A. EpiOcular™ (MatTek)
B. SkinEthic™ HCE (L’Oréal)
C. BCOP – Bovine Corneal Opacity/Permeability
D. LabCyte Cornea-MODEL
Correct Answer: C (BCOP is an excised animal tissue test, not an RhCE. Validated RhCE models include EpiOcular™, SkinEthic HCE, LabCyte HCE, and MCTT HCE, all of which are human cell-derived corneal epithelium constructs) .
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Q4. What is an advantage of using the OECD 492 RhCE tests in a testing strategy?
A. They can over-predict skin sensitization
B. They allow identification of non-irritants without animal testing, thus reducing live animal use in ocular safety testing .
C. They are cheaper than all in chemico methods
D. They can distinguish between reversible and irreversible eye damage on their own
Correct Answer: B (RhCE assays are animal-free and have been validated to reliably flag chemicals that are not eye irritants, which helps in waiving in vivo tests for those substances and reduces animal testing. They are commonly used in a bottom-up approach: if a substance is predicted as non-irritant by RhCE, no rabbit test is needed) .
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Q5. True or False: OECD TG 492 can be used as a full replacement to classify chemicals into all UN GHS eye hazard categories (1, 2, or no category).
Correct Answer: False. (The original OECD 492 is validated for identifying “No Category” chemicals (non-irritants). It cannot by itself differentiate between Category 1 (serious damage) and Category 2 (irritant) – a chemical that reduces viability below the threshold is just “irritant or worse” and would need further testing (like BCOP/ICE or a defined approach) to determine if it’s Cat 2 or Cat 1. However, a newer RhCE-based test method (e.g. OECD 492B SkinEthic™) and Defined Approaches now allow full classification with combinations.)
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OECD TG 497 – Defined Approaches for Eye Irritation and Serious Eye Damage
Q1. What is meant by “Defined Approaches (DAs) for Eye Irritation/Serious Eye Damage” in OECD TG 497 (and the updated TG 467 for eye)?
A. A probabilistic weight-of-evidence narrative
B. A fixed, rule-based integration of multiple test results (in vitro assays and/or physicochemical data) to derive a hazard classification for eye irritation (GHS Cat 1, 2, or No Cat)
C. A single new in vivo rabbit test
D. An approach that defines the eye structure in detail
Correct Answer: B (Defined Approaches are like “testing strategies” that have predefined decision logic using results from several non-animal methods. For eye irritation, DAs might combine, for example, BCOP, RhCE, and physicochemical properties in an algorithm to directly output a GHS category) .
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Q2. According to OECD’s defined approaches for eye hazard, which of the following sets of methods might be used together to fully replace the Draize eye test for liquids?
A. Skin sensitization assays only
B. Physicochemical property (like pH) + BCOP (OECD 437) + RhCE test (OECD 492) in an integrated decision tree (This describes DA for liquids – “DAL1”) .
C. Acute oral toxicity + BCOP
D. Just one RhCE test
Correct Answer: B (One of the defined approaches for eye irritation is DAL1, which uses the substance’s physical/chemical properties, plus results from a BCOP opacity measurement and a human corneal epithelium model test. Another DA, DAL2, uses BCOP and the Short Time Exposure test (OECD 491) for liquids. These combinations have defined rules to classify a chemical as Cat 1, Cat 2, or No Cat) .
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Q3. What is an example of how a Defined Approach (DA) might classify a chemical as “No Category” (non-irritant) for eyes?
A. If it has very low pH
B. If in a DA (say, for a solid), BCOP gives a low IVIS and RhCE viability is high, meeting the DA criteria for No Cat .
C. If any single test is negative, automatically
D. It cannot classify No Cat
Correct Answer: B (For instance, the DA for solids (DAS) might say: if BCOP IVIS is below the Cat 1 threshold and RhCE indicates no irritation, then the substance can be classified as No Cat. Each DA has rules; generally a combination of negative/non-irritant outcomes in component assays leads to an overall “No Cat” classification) .
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Q4. True or False: The Defined Approaches in OECD TG 497/467 allow for distinguishing between GHS Category 1 and Category 2 without animal testing.
Correct Answer: True. (The DAs are designed to fully replace the Draize test, meaning they can identify Category 1, Category 2, and No Cat. For example, if BCOP indicates severe damage (Cat 1) in a DA, it will conclude Cat 1; if BCOP is not Cat 1 but RhCE/STE show irritation, the DA can conclude Cat 2. Thus, using the combination of tests and decision rules, one can assign the correct category in many cases) .
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Q5. What is the main benefit of using an OECD 497 Defined Approach for eye hazard over a single in vitro test?
A. It’s cheaper than any single test (not necessarily true)
B. It improves predictive accuracy by covering multiple mechanisms/endpoints, thereby compensating for limitations of individual tests and providing a confident classification for regulatory purposes .
C. It requires no expertise to interpret
D. It eliminates all testing needs
Correct Answer: B (Each in vitro test has an applicability domain and certain false negatives/positives. By combining tests (e.g., one that’s good at identifying Cat 1 and another good at identifying No Cat), the DA yields a more robust prediction. The defined approach provides a clear, rule-based outcome that regulators can accept in lieu of an animal test, improving overall prediction and adhering to 3Rs.)
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OECD TG 408: Repeated Dose 90-Day Oral Toxicity in Rodents
Question: Which of the following is NOT evaluated by an OECD TG 408 90-day oral toxicity study?
A. Chronic effects such as carcinogenicity developing after prolonged exposure
B. Identification of target organ toxicity from subchronic exposure
C. Clinical pathology (e.g. blood chemistry) changes in treated rodents
D. Determination of a No-Observed-Adverse-Effect Level (NOAEL)
Correct Answer: A
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Question: One limitation of the 90-day oral toxicity test (TG 408) is that it:
A. cannot detect reproductive or developmental toxicity that would require separate studies
B. fails to identify any target organ toxicity in rodents
C. uses only male animals, missing female-specific effects
D. cannot establish a dose–response for observed toxic effects
Correct Answer: A
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Question: A 90-day study in rats showed no adverse effects up to the limit dose. Why might a follow-up chronic study still be considered?
A. Some toxic effects (e.g. tumor formation) may require longer exposure than 90 days to manifest
B. Regulatory guidelines always require a 2-year study regardless of 90-day results
C. The 90-day study cannot establish a NOAEL for risk assessment purposes
D. The subchronic study is invalid if it shows no effects at the limit dose
Correct Answer: A
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Question: Data from an OECD 408 study are often used for all EXCEPT which of the following?
A. Setting exposure limits based on the subchronic NOAEL
B. Identifying specific target organs affected by the test substance
C. Classifying a chemical for long-term toxicity (STOT-RE) under GHS criteria
D. Determining a chemical’s potential to induce developmental malformations
Correct Answer: D
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OECD TG 414: Prenatal Developmental Toxicity Study
Question: The OECD 414 prenatal developmental toxicity test is primarily designed to detect:
A. Structural malformations and embryo-fetal lethality during prenatal development
B. Learning and memory deficits in offspring after birth
C. Effects on male and female fertility across generations
D. Immune function alterations in adult offspring
Correct Answer: A
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Question: Which outcome cannot be assessed in a standard OECD 414 study?
A. Postnatal cognitive or behavioral defects in offspring (e.g. learning delays)
B. Fetal skeletal variations and malformations at term
C. Embryo implantation loss or resorptions during pregnancy
D. Maternal toxicity manifesting as reduced body weight gain during gestation
Correct Answer: A
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Question: In an OECD 414 study, a test chemical caused fetal malformations only at a dose that also caused severe maternal toxicity. What is a key interpretation challenge in this scenario?
A. Determining if the fetal effects are secondary to maternal toxicity or indicative of true developmental toxicity
B. Concluding that the chemical is not teratogenic since maternal toxicity occurred
C. Classifying the chemical as a definitive teratogen (Category 1) despite confounding maternal effects
D. Ignoring maternal health effects when assessing developmental toxicity
Correct Answer: A
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Question: Which statement about the OECD 414 developmental toxicity guideline is TRUE?
A. It typically includes testing in two species (one rodent and one non-rodent) to account for species differences
B. It evaluates offspring for functional deficits through adulthood
C. It assesses fertility parameters in the F1 generation by mating the offspring
D. It can identify endocrine-disrupting effects on postnatal development without additional tests
Correct Answer: A
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OECD TG 420: Acute Oral Toxicity – Fixed Dose Procedure
Question: What is a key limitation of the OECD 420 Fixed Dose Procedure for acute oral toxicity compared to traditional LD50 tests?
A. It does not determine an exact LD50 value for the test substance
B. It requires a larger number of animals than the classical LD50 test
C. It cannot be used to classify substances into GHS acute toxicity categories
D. It provides no information on clinical signs of toxicity in test animals
Correct Answer: A
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Question: The Fixed Dose Procedure (TG 420) for acute toxicity is characterized by:
A. Identifying a dose that causes evident but sub-lethal toxicity signs to assign hazard category
B. Dosing animals at many incremental levels to pinpoint the LD50 with precision
C. Using in vitro cell assays instead of live animals to determine acute toxicity
D. Failing to produce any information about symptoms of overdose in animals
Correct Answer: A
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Question: Which statement about the outcome of an OECD 420 acute oral toxicity test is CORRECT?
A. It allows assignment of a GHS acute toxicity category based on presence or absence of evident toxicity at certain fixed doses
B. A “negative” result (no toxicity at the limit dose) means the substance is completely non-toxic in humans
C. If no animals die at the highest fixed dose tested, the LD50 is considered to be zero
D. The Fixed Dose Procedure provides a more precise toxicity threshold than any other acute test method
Correct Answer: A
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OECD TG 432: In Vitro 3T3 NRU Phototoxicity Test
Question: Which of the following is a known limitation of the 3T3 Neutral Red Uptake Phototoxicity test (OECD 432)?
A. It cannot detect phototoxic chemicals that require metabolic activation (pro-photens) to become reactive
B. It has a high false-negative rate for known photoirritant chemicals that act directly
C. It relies on live animals to assess phototoxic potential of chemicals
D. It can only test chemicals that do not absorb UV or visible light
Correct Answer: A
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Question: The OECD 432 phototoxicity assay primarily identifies substances that:
A. Cause increased cell death in vitro when exposed to light (photo-cytotoxicity)
B. Cause immune-mediated allergic skin reactions upon light exposure (photoallergy)
C. Induce mutations in bacteria under UV light exposure
D. Cause eye irritation upon exposure to UV light
Correct Answer: A
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Question: A chemical tested negative in the 3T3 NRU phototoxicity test. In which case should there still be concern about phototoxic risk?
A. The chemical is a “pro-phototoxin” that needs metabolic conversion in skin to a phototoxic form (not captured in the 3T3 assay)
B. The chemical strongly absorbs UVA light and was positive in the assay
C. The chemical does not absorb any UV/visible light energy
D. The chemical was negative and has no structural similarity to known phototoxins
Correct Answer: A
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Question: Which class of substances might produce a false positive in the in vitro phototoxicity test?
A. Strong oxidizing agents that can non-specifically damage the test cells under light exposure (even if they may not cause phototoxicity in vivo)
B. Known human phototoxins such as psoralens (which the assay is designed to detect)
C. Substances that require systemic metabolism to become photoreactive (pro-phototoxins)
D. Substances that do not absorb light in the UVA/visible range
Correct Answer: A
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OECD TG 437: Bovine Corneal Opacity and Permeability (BCOP) Test
Question: The BCOP eye irritation test has a known limitation in that it cannot measure:
A. Injury to conjunctiva and iris (redness or iritis) that would occur in a living eye
B. Corneal opacity and permeability changes caused by corrosive chemicals
C. Severe irreversible damage to the cornea from strong irritants
D. The combined effects of opacity and epithelial damage in an isolated cornea
Correct Answer: A
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Question: Which of the following is TRUE regarding the limitations of OECD TG 437 (BCOP)?
A. It tends to overpredict the eye irritation hazard of certain alcohols and ketones (false positives)
B. It has a high false-negative rate for substances that cause severe corneal injury
C. It can distinguish mild (reversible) irritants from non-irritants with high accuracy
D. Positive results in BCOP are not accepted for regulatory classification of eye irritants
Correct Answer: A
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Question: In the BCOP test, a high In Vitro Irritancy Score (IVIS) indicates:
A. The test substance is likely to cause serious eye damage (UN GHS Category 1) without further testing
B. The test substance is a mild eye irritant that requires in vivo confirmation
C. The substance is not an eye irritant (No Category)
D. Inconclusive results, since BCOP cannot identify severe irritants
Correct Answer: A
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Question: A chemical tested in BCOP showed very low opacity and permeability (IVIS below the cutoff for Category 1). What is the appropriate interpretation?
A. The chemical is not predicted to cause serious eye damage, but it could still cause moderate irritation (Category 2) and may need additional testing
B. The chemical can be classified as “non-irritating to eyes” with no further evidence
C. The BCOP result confirms the chemical is safe for eye contact in humans
D. The chemical should be immediately tested in a Draize rabbit eye test to verify non-irritancy
Correct Answer: A
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OECD TG 438: Isolated Chicken Eye (ICE) Test
Question: What is a known limitation of the Isolated Chicken Eye test (OECD 438)?
A. It has a high false-negative rate for certain solids and surfactant-based chemicals that cause irritation in vivo
B. It measures conjunctival redness and swelling, which can lead to over-prediction of mild irritants
C. It requires live chickens to be dosed over several days
D. It cannot detect severe ocular corrosives (UN GHS Cat. 1)
Correct Answer: A
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Question: The ICE test is not recommended for distinguishing substances that:
A. cause only moderate, reversible eye irritation (Category 2) from those that are non-irritants
B. cause serious eye damage (Category 1) from those that are non-irritating
C. are expected to be non-irritants, as ICE inherently over-predicts hazard
D. are liquids or water-soluble, as the test applies only to solids
Correct Answer: A
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Question: Both the BCOP (TG 437) and ICE (TG 438) assays share which limitation?
A. They cannot assess damage to conjunctiva or iris, nor the recovery (reversibility) of eye lesions over time
B. They cannot detect chemicals that cause severe eye corrosion (Category 1)
C. They cannot be used on mixtures or formulations
D. They tend to underpredict the hazard of all alcohols and ketones
Correct Answer: A
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Question: In an ICE test, a chemical does not induce significant corneal opacity or swelling (thus not classified as Cat.1). What is the next step regarding eye irritation classification?
A. Recognize that the chemical could still be an irritant (Cat.2) and consider additional testing (e.g. a RhCE test or in vivo study) to determine if it causes moderate irritation
B. Conclude the chemical is not an eye irritant at all and no further testing is needed
C. Classify the chemical as “No Category” for eye irritation based on ICE alone
D. Repeat the ICE test to confirm the negative result before any further action
Correct Answer: A
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OECD TG 442A: Skin Sensitization – Local Lymph Node Assay: DA (ATP assay)
Question: The LLNA: DA (OECD 442A) is a non-radioactive LLNA that measures ATP content. For which type of test substance is the LLNA: DA not appropriate?
A. Substances that drastically alter ATP levels or contain ATP-degrading enzymes, confounding the assay readout
B. Potent organic sensitizers that cause strong lymph node responses
C. Common cosmetic ingredients like fragrances (small organic molecules)
D. Test substances that are soluble and not overly irritant on application
Correct Answer: A
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Question: Which limitation of the traditional LLNA (OECD 429) also applies to the modified LLNA: DA (442A)?
A. Certain metal allergens (e.g. nickel compounds) may yield false negatives due to poor detection in the mouse LLNA model
B. It requires the use of radioactive isotopes for measuring cell proliferation
C. It cannot be used for regulatory classification of skin sensitizers
D. It cannot distinguish between irritants and sensitizers under any circumstances
Correct Answer: A
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Question: Strong skin irritants that are not true sensitizers can cause what issue in an LLNA (including 442A)?
A. A false positive result – the irritant inflammation can stimulate lymph node proliferation resembling a sensitization response
B. A false negative result – irritant effects suppress the immune response and mask sensitization
C. No lymph node activation at all – irritants do not affect lymph nodes in the LLNA
D. Selective activation of only T-cells without B-cell involvement
Correct Answer: A
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Question: In which situation might a guinea pig test (TG 406) be considered instead of any LLNA (including 442A)?
A. When the test substance is a metal or other material not reliably detected by the mouse LLNA methods
B. When a quicker, less expensive screening is needed over the LLNA
C. When only a small amount of test substance is available
D. When avoiding animal testing altogether (to use an in vitro method)
Correct Answer: A
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OECD TG 442B: Skin Sensitization – Local Lymph Node Assay: BrdU-ELISA
Question: The LLNA: BrdU-ELISA (OECD 442B) measures lymphocyte proliferation via BrdU incorporation. Which substance could interfere with this assay’s accuracy?
A. A cytostatic chemical that inhibits DNA synthesis in lymphocytes (potentially causing a false negative result)
B. A moderate sensitizer that causes lymphocyte proliferation (the assay is intended to detect this)
C. A radioactive compound in the test substance (radiolabeling is not used in BrdU-ELISA)
D. An ATP synthase inhibitor (this specifically impacts LLNA: DA more than BrdU)
Correct Answer: A
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Question: The non-radioactive LLNA methods (442A and 442B) share which limitation with the traditional LLNA?
A. They may not accurately detect certain classes of sensitizers like heavy metals, which often yield false negatives in LLNA models
B. They cannot distinguish strong sensitizers from weak sensitizers
C. They have completely eliminated false positives from skin irritants
D. They are not accepted for regulatory hazard classification of skin sensitizers
Correct Answer: A
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Question: In the BrdU-ELISA LLNA (442B), what result might a strong irritant (that is not an allergen) produce?
A. An elevated BrdU incorporation (false positive indicating “sensitization”) due to non-specific cell proliferation from irritation
B. No lymph node cell proliferation at any dose (clear negative, correctly indicating no sensitization)
C. A decrease in BrdU incorporation relative to control
D. It cannot be determined – irritants are excluded from testing in LLNA
Correct Answer: A
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Question: When might the guinea pig maximization test or Buehler test (OECD 406) be preferable over LLNA: BrdU-ELISA (442B)?
A. If the test substance is highly insoluble or a known immunosuppressant that the LLNA might not detect reliably
B. If a faster result is needed, since guinea pig tests are quicker than LLNA
C. If the substance is a strong sensitizer, because LLNA cannot detect strong sensitizers
D. If the goal is to reduce animal use, since guinea pig tests use fewer animals than LLNA
Correct Answer: A
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OECD TG 442C: In Chemico Skin Sensitisation – Direct Peptide Reactivity Assay (DPRA)
Question: Which statement about the Direct Peptide Reactivity Assay (DPRA, OECD 442C) is CORRECT?
A. It measures a chemical’s reactivity with model peptides to predict the skin sensitization molecular initiating event (protein binding)
B. It uses living immune cells to detect dendritic cell activation markers
C. It can detect chemicals that require metabolic activation (pro-haptens) to become sensitizers
D. It alone provides definitive skin sensitization classification including potency sub-categories
Correct Answer: A
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Question: The DPRA is unable to detect some sensitizers. Which scenario represents a known limitation of this assay?
A. A test chemical needs enzymatic activation in the skin to bind proteins (pro-hapten), so it shows no peptide reactivity in DPRA (false negative)
B. A test chemical directly binds to cysteine or lysine peptides, showing depletion in DPRA
C. A highly reactive organic acid that depletes peptide in DPRA and is a strong sensitizer in vivo
D. A test chemical that is water-soluble and stable – DPRA yields a negative result
Correct Answer: A
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Question: Which class of substances might cause a false positive in the DPRA (peptide reactivity assay)?
A. Strong oxidizers or substances that non-specifically degrade or modify the peptides (e.g. bleach), even if they may not cause sensitization in vivo
B. Pro-haptens that require metabolism (these usually cause false negatives, not positives, in DPRA)
C. Heavy metal salts (these often do not show peptide binding in DPRA)
D. Non-reactive substances that do not form adducts with the peptide (would be correctly negative, not false positive)
Correct Answer: A
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Question: For which of the following should the DPRA not be used as a stand-alone test?
A. Metal compounds and inorganic complexes that do not form covalent bonds with the model peptides
B. Organic electrophiles that directly modify cysteine or lysine residues
C. A small fragrance molecule known to cause contact allergy via protein binding
D. A mixture of low-molecular-weight organic sensitizers
Correct Answer: A
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Question: If a chemical tests negative in the DPRA, what is the best next step in an integrated skin sensitization testing strategy?
A. Evaluate the chemical with additional sensitization assays (e.g. KeratinoSens and/or h-CLAT) to ensure it’s not a sensitizer missed by DPRA
B. Declare the chemical a non-sensitizer and forego any further testing
C. Perform a repeat DPRA to confirm the negative result, as no other assays are needed
D. Classify the chemical as “No GHS Category” for skin sensitization based on DPRA alone
Correct Answer: A
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OECD TG 442D: In Vitro Skin Sensitisation – KeratinoSens™ (ARE-Nrf2 Luciferase Test)
Question: Which situation is likely to lead to a false negative result in the KeratinoSens assay (OECD 442D)?
A. The test chemical requires metabolic activation (a pro-hapten) or slow oxidation to become reactive, which the keratinocyte assay cannot adequately provide
B. The test chemical is a strong electrophile that directly activates the Nrf2 pathway
C. The test chemical causes general oxidative stress in keratinocytes
D. The test chemical is soluble and stable under the test conditions
Correct Answer: A
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Question: A known limitation of the KeratinoSens™ assay is that it:
A. may not detect sensitizers that exclusively react with lysine residues or that need metabolism (leading to possible false negatives)
B. relies on animal test data to confirm each positive result
C. tends to produce false negatives for all strong sensitizers (potent sensitizers are usually caught, not missed)
D. measures only peptide binding to predict sensitization potential
Correct Answer: A
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Question: A positive result in the OECD 442D KeratinoSens assay indicates that a substance:
A. Activates the antioxidant/electrophile response element (ARE-Nrf2) pathway in vitro, consistent with potential skin sensitizing activity
B. Will definitively cause allergic contact dermatitis in humans upon exposure
C. Should be classified immediately as a skin sensitizer without need for any other information
D. Activates T-cells directly in an immune assay
Correct Answer: A
116
Question: KeratinoSens may yield a false positive for a test chemical that:
A. Causes generic cellular stress or cytotoxicity in keratinocytes (e.g. a non-sensitizing irritant that still triggers the Nrf2 pathway)
B. Requires metabolic activation to form a protein-reactive metabolite
C. Is highly hydrophobic (log P > 7), preventing adequate exposure to the cells
D. Does not induce any oxidative stress in the cells
Correct Answer: A
117
OECD TG 442E: In Vitro Skin Sensitisation – human Cell Line Activation Test (h-CLAT)
Question: What is a known applicability limitation of the h-CLAT assay (OECD 442E)?
A. Test chemicals with log P > 3.5 or very weak sensitizers may give false-negative results due to poor cellular uptake or low response
B. It cannot correctly identify strong sensitizers (UN GHS Cat.1A)
C. It produces no false positives with irritant-only substances
D. It requires the use of radioactive reagents to label antibodies
Correct Answer: A
118
Question: Which property of a test substance could interfere with obtaining a reliable result in the h-CLAT?
A. The substance is highly autofluorescent at the flow cytometry detection wavelengths, masking the detection of CD86/CD54 markers
B. The substance is highly water-soluble and easily penetrates cells
C. The substance is a moderate sensitizer that upregulates CD54 and CD86
D. The substance has a log P around 1–2, indicating good bioavailability in the assay
Correct Answer: A
119
Question: An h-CLAT test yields a negative result for a chemical that is relatively lipophilic (log P ~4). How should this outcome be interpreted?
A. With caution – the chemical’s lipophilicity may limit its availability to the cells, so a false negative is possible (additional evidence or tests should be considered)
B. As a confirmed non-sensitizer – no sensitization potential since h-CLAT was negative
C. As a false positive – lipophilic substances are known to always give positive results in h-CLAT
D. As an indicator that the chemical likely works through a different key event (protein binding) and is still a sensitizer
Correct Answer: A
120
Question: Which of the following is TRUE about the h-CLAT method?
A. It measures changes in human monocytic cell surface markers (like CD86 and CD54) to indicate dendritic cell activation, but doesn’t directly measure T-cell response
B. It can on its own distinguish skin sensitizer potency (whether Cat.1A vs 1B) with high accuracy
C. It is not accepted by regulatory authorities for any decision-making
D. It detects protein binding of chemicals using a peptide depletion approach
Correct Answer: A
121
OECD TG 443: Extended One-Generation Reproductive Toxicity Study (EOGRTS)
Question: Which potential effect on offspring would not be evaluated in an OECD 443 study unless an additional specialized cohort is included?
A. Neurobehavioral development and cognitive function of offspring (developmental neurotoxicity)
B. Growth, survival, and morphology of offspring from birth through weaning
C. Reproductive performance (fertility) of the parental (F0) generation
D. Physical development and attainment of puberty in F1 offspring
Correct Answer: A
122
Question: If an OECD 443 study is conducted without producing a second filial generation (no F2), what is a resulting limitation?
A. The fertility of the F1 offspring is not directly confirmed (since they are not bred to produce an F2)
B. The fertility of the F0 parents cannot be assessed in the absence of an F2
C. No evaluation of developmental toxicity is possible in an EOGRTS
D. Potential effects on the F1 generation cannot be observed at all
Correct Answer: A
123
Question: What additional endpoints can OECD 443 include to broaden its scope beyond a standard reproductive study?
A. It can incorporate dedicated cohorts to assess developmental neurotoxicity or immunotoxicity in offspring, which are otherwise not examined by default
B. It includes full lifetime carcinogenicity assessment in the offspring
C. It measures behavioral teratology and adult cognitive performance in F2 animals by default
D. It evaluates transgenerational genetic effects (F3 generation) as part of the core study design
Correct Answer: A
124
Question: A chemical tested in an EOGRTS (TG 443) caused reduced litter sizes and delayed development of pups. For regulatory purposes, this substance would likely be:
A. Classified as a reproductive/developmental toxicant (e.g. GHS Reproductive Toxicity Category 2 or 1)
B. Classified only after a second-generation test confirms the effect
C. Considered non-hazardous to reproduction because only one generation was tested
D. Retested in a prenatal developmental toxicity study (TG 414) before any classification
Correct Answer: A
125
OECD TG 471: Bacterial Reverse Mutation Test (Ames test)
Question: The Ames test (OECD 471) has a well-known limitation in that it cannot detect:
A. Chromosome damage or aneuploidy (chromosomal aberrations) caused by a test substance
B. Gene mutations resulting from base-pair substitutions
C. Gene mutations resulting from frameshift events
D. Point mutations in bacterial DNA
Correct Answer: A
126
Question: Which of the following is a reason that a mutagenic chemical might yield a false negative in an OECD 471 test?
A. The chemical requires mammalian metabolism or specific conditions not provided by the standard bacterial assay (thus it shows no mutagenicity in bacteria)
B. The chemical is a direct-acting mutagen that readily causes reversions in tester strains
C. The S9 metabolic activation mix is included, leading to over-estimation of mutagenic potential
D. The chemical causes base-pair substitutions, which the Ames strains cannot detect
Correct Answer: A
127
Question: A test compound is a known clastogen (causes chromosome breaks) but is not a point mutagen. What result would you expect in the OECD 471 Ames test, and why?
A. A negative Ames test result, because the compound’s genotoxicity is through chromosomal damage rather than point mutations (which Ames cannot detect)
B. A positive Ames test result, because any genotoxic mechanism will produce a positive in Ames
C. An equivocal result, because chromosome breakers always yield suspicious colony growth patterns in Ames
D. A false positive Ames result, because clastogens kill bacteria and cause them to mutate
Correct Answer: A
128
Question: Why does a negative result in an Ames test not guarantee that a chemical is non-carcinogenic?
A. The chemical could cause cancer via non-genotoxic mechanisms or via genetic damage not detectable in bacteria (e.g. chromosomal damage in mammals)
B. The Ames test has a high false-positive rate, not a false-negative issue
C. Bacteria have different DNA repair processes that make the Ames test overly protective
D. A single test is sufficient – a negative Ames actually does guarantee non-carcinogenicity
Correct Answer: A
129
Question: In regulatory genotoxicity testing, the Ames test is usually paired with other assays primarily because:
A. It only detects point mutations, so additional tests (for chromosomal damage, e.g. micronucleus or chromosomal aberration assays) are needed to catch other genotoxic mechanisms
B. It is too expensive to run as a stand-alone test without corroboration
C. It often produces false negatives for strong mutagens and needs confirmation by identical repeat tests
D. It cannot be conducted on chemicals that are volatile or insoluble
Correct Answer: A
130
OECD TG 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Eye Irritation
Question: OECD TG 492 RhCE assays (e.g. EpiOcular™) are primarily used to:
A. Identify chemicals that do not require classification for eye irritation (i.e. non-irritants)
B. Directly classify chemicals as UN GHS Category 1 (serious eye damage)
C. Determine if eye irritation effects are reversible or not
D. Measure conjunctival redness and swelling in vitro
Correct Answer: A
131
Question: What is a limitation of the RhCE in vitro eye irritation test (OECD 492)?
A. It cannot evaluate injuries to conjunctival or iris tissue, nor predict if corneal damage would be reversible in vivo
B. It fails to detect when a substance is non-irritating, resulting in many false negatives for non-irritants
C. It cannot be used on water-soluble materials
D. It over-predicts eye irritation for all types of surfactants and alcohols
Correct Answer: A
132
Question: A chemical yields a positive result in an OECD 492 RhCE test. How should this result be interpreted for classification?
A. The substance is considered to cause eye irritation – it should be classified at## OECD TG 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Eye Irritation
Question: OECD TG 492 RhCE assays (e.g. EpiOcular™) are primarily used to:
A. Identify chemicals that do not require classification for eye irritation (i.e. non-irritants)
B. Directly classify chemicals as UN GHS Category 1 (serious eye damage)
C. Determine if eye irritation effects are reversible or not
D. Measure conjunctival redness and swelling in vitro
Correct Answer: A
133
Question: What is a limitation of the RhCE in vitro eye irritation test (OECD 492)?
A. It cannot evaluate injuries to conjunctival or iris tissue, nor predict if corneal damage would be reversible in vivo
B. It fails to detect when a substance is non-irritating, resulting in many false negatives for non-irritants
C. It cannot be used on water-soluble materials
D. It over-predicts eye irritation for all types of surfactants and alcohols
Correct Answer: A
134
Question: A chemical yields a positive result in an OECD 492 RhCE test. How should this outcome be interpreted?
A. The substance is considered to cause eye irritation; it should be classified at least as an eye irritant (Category 2), while further information (e.g. additional in vitro tests or weight-of-evidence) is needed to determine if it could cause serious eye damage (Category 1)
B. The substance is confirmed as non-irritating to eyes, since TG 492 is only used to rule out irritants
C. A positive RhCE result alone definitively classifies a substance as causing irreversible eye damage (Category 1)
D. The result is not useful for regulatory purposes, so an in vivo rabbit eye test must be conducted for classification
Correct Answer: A
135
Question: Which statement correctly compares the BCOP, ICE, and RhCE in vitro eye irritation tests?
A. BCOP (TG 437) and ICE (TG 438) are suitable for identifying chemicals causing serious eye damage (Cat. 1), whereas the RhCE test (TG 492) is mainly used to identify non-irritants (chemicals not requiring eye irritation labeling)
B. BCOP and ICE can on their own reliably distinguish moderate irritants (Cat. 2) from non-irritants without additional tests
C. A positive result in any of these tests (BCOP, ICE, or RhCE) directly indicates irreversible eye damage in vivo
D. The BCOP and ICE methods include assessment of conjunctival redness and iris lesions, unlike the RhCE method
Correct Answer: A
136
OECD TG 497: Defined Approaches on Skin Sensitisation
Question: What is an OECD Defined Approach for skin sensitisation (Guideline 497)?
A. A fixed integrated testing strategy that combines multiple non-animal information sources (in chemico, in vitro, in silico) with a defined data interpretation procedure to predict skin sensitization hazard
B. A single test method using a human cell line to identify skin sensitizers
C. An in vivo assay using fewer animals than the LLNA
D. A weight-of-evidence approach with no predefined rules for interpretation
Correct Answer: A
137
Question: Which of the following is a benefit of using a Defined Approach (TG 497) compared to individual sensitization assays?
A. It can overcome some limitations of any one method by combining results, improving overall predictive performance for sensitization hazard
B. It requires only one assay to make a decision, simplifying testing
C. It eliminates the need for understanding mechanistic key events in skin sensitization
D. It guarantees 100% accuracy in identifying human skin sensitizers
Correct Answer: A
138
Question: If two out of three component assays in a skin sensitization Defined Approach (e.g. DPRA, KeratinoSens™, h-CLAT) are positive for a chemical, the Defined Approach prediction would most likely:
A. Classify the chemical as a skin sensitizer (since a majority of the assays indicate sensitization)
B. Classify the chemical as a non-sensitizer (because not all tests were positive)
C. Be “inconclusive” and automatically require an animal test
D. Override the results and default to the LLNA outcome
Correct Answer: A
139
Question: One challenge when applying OECD 497 Defined Approaches in regulation is:
A. Ensuring that if a chemical falls outside the applicability domain of one or more component assays (e.g. extremely volatile, insoluble, or requiring metabolic activation), the integrated prediction is interpreted with caution or may be considered inconclusive
B. That the Defined Approach requires mandatory animal testing to confirm any prediction
C. Lack of any regulatory acceptance for Defined Approach results in skin sensitization
D. The inability of Defined Approaches to provide information on sensitizer potency (sub-categorization)
Correct Answer: A
