Domande esame Biotechnologies: experimental model in research

Caloni.

Io ho svolto l’esame nella sessione estiva del 2024, pertanto le informazioni sono aggiornate alla tale

data.

1) Phototoxicity: in vitro validated test

Phototoxicity, or photoirritation, is an acute skin reaction related to non-immunological xenobiotic

exposure in the presence of light, particularly in the UVA range (320-400 nm). It is caused by

simultaneous exposure to xenobiotic and light. The OECD has not yet accepted a guideline for

photoirritation in vivo but recommended those in vitro.

Tier approach:

- 3T3 NRU: Phototoxicity test

Workflow:

1. Treatment with the test substance

2. Irradiation

3. Neutral Red Uptake a say —> viability test

It is predictive for photosensitizing and non-photosensitizing substances, but it is limited for

water-soluble test substances.

The Neutral Red Uptake assay indicates that if the cells are red, they have retained the red

compound in their lysosomes (they are alive). On the contrary, if they are pink, they are dead.

- RhE PT test = Reconstructed human Epidermis Phototoxicity Test

The test chemical is applied topically to a three-dimensional (3D) RhE tissue composed of human-

derived keratinocytes.

This test is valid also for complex mixtures and pharmacological patch, and being 3D allow

histological evaluation.

Moreover, it can test water soluble substances.

Endpoint = cell viability —> MTT test

2) IATA definition

IATA means Integrated Approach for Testing and Assestment.

It includes:

- In vitro

- In vivo 1 Alternative Methods in Toxicology – domande e risposte

- In silico

- In chemico

- Omic technologies

3) What is meant for “validation”?

Validation is meant an approach that allows identifying the Reliability, Reproducibility, Relevance

and Predictivity of a method.

To validate an Alternative Method is mandatory to follow 5 steps:

1. Development of the test

It’s important to establish the Field of Application, the Test Protocol and the Prediction

Model.

2. Pre-validation

Improve the Test Protocol and the Prediction Model

▪ Inter-laboratory comparison

▪

3. Validation

Interlaboratory test under Blind Conditions (EU-NETVAL, Network of lab)

▪ Relevance and Reliability for the Field of Application designed

▪

4. Independent evaluation (Peer review) —> ESAC (Expert Scientific Advisory Committee)

5. Official approval —> OECD guideline

The validation studies can be:

- IN PERSPECTIVE = data generated through collaborative studies between different labs

- RETROSPECTIVE = existing data produced by the inventor during development

- CATCH UP/ME-TOO = data from similar methods

4) Skin irritation and Alternative Method

The skin irritation is a local inflammation of non-immunological origin, characterized by erythema,

edema or necrosis due to single or repeated exposure to a substance (e.g., HCl or HF).

An irritating potential can cause effects on the stratum corneum, epidermis and dermis.

The most important characteristics is the REVERSIBILITY.

Alternative methods that can be used instead of the Draize test (in vivo test on albino rabbits) include:

Reconstructed Human Epidermis (RhE) models:

• EPISKIM

• EPIDERM TG 439

• SKINETHIC

The endpoint for the 3 tests is the cell viability/cytotoxicity analyzed using MTT test (from yellow

tetrazolium salt to a blue formazan salt):

≤

- Cell viability 50% —> IRRITANT 2 Alternative Methods in Toxicology – domande e risposte

- Cell viability > 50% —> NON-IRRITANT

Episkin analyses also the release of interleukin-1alpha

These tests allow to differentiate between different chemicals, in particular allowing to differentiate

between substances not classifiable by irritation (No cat.) and irritants (Cat.2) but not for week irritant

(Cat.1).

Moreover, they are not useful for aerosol and gaseous substances.

5) OECD definition

OECD stands for Organization for Economic Cooperation and Development.

6) TG definition

TG stands for Test Guideline.

7) BCOP

BCOP is an acronym for Bovine Cornea and Permeability test.

It’s an EX VIVO model that allows to study the eye irritation, which involves ocular changes following

the application of test substance ON THE FRONT SURFACE of the eye, totally reversible within 21

days from application.

BCOP helps analyze chemicals inducing severe damage and identify those not requiring

classification for eye irritation or serious eye damage.

The endpoints are: Corneal Opacity (decrease in light transmission) and Corneal Permeability

(fluorescein).

The In Vitro Irritancy Score (IVIS) ranges:

0 - 3 Non-irritants

3,1 - 25 Weak irritant

25,1 - 55 Moderate irritant

>55,1 Severe/corrosive irritant

8) Internal Dose Concept

The internal dose concept refers to the free concentration able to reach the cellular target.

3 Alternative Methods in Toxicology – domande e risposte

9) mEST

mEST stands for “Murine Embryonic Stem Cell Test” and is an alternative method used to determine

the embryotoxicity of a substance. It uses D3 cells (undifferentiated embryonic cells) and BALB-3T3

fibroblasts (adult cells).

The two lines are incubated with potential embryotoxicity substance for 10 days, testing both

inhibition of differentiation and proliferation.

Procedure:

- Day 0= hanging drops

- Day 1-3= aggregation of stem cells

- Day 3-5= embryoid bodies

- Day 5-10= contracting cardiomyocytes

The endpoints are:

- Cytotoxicity D3 analyzed through MTT test —> IC D3

50

- Cytotoxicity BALB/3T3 analyzed through MTT test —> IC 3T3

50

- Developmental toxicity (cardiomyocytes) —> ID

50

In this way is possible to say if a substance is NO EMBRYOTOXIC, WEAK EMBRYOTOXIC or STRONG

EMBRYOTOXIC.

10) NAM definition

NAM can mean:

- New Alternative Methodologies

- Non-Animal Method

11) ITS definition

ITS stand for Integrated Testing Strategy.

12) Skin corrosion and Alternative Methods

Skin corrosion is an irreversible skin damage due to the exposure to an external xenobiotic. Different

substances, including chemicals and cosmetics, can be corrosive.

Alternative Methods available today include:

- TER = Trans Epithelial Electrical Resistance (TG 430)

It’s an ex vivo method that uses rat skin to differentiate between corrosive and non-

corrosive substances. The endpoint is the alteration of the skin barrier.

However, it is not useful for gaseous and areosol substances.

- EPISKIN/ EPIDERM/ SKINETHIC/ EPISC = RhE Model test (TG 431)

4 Alternative Methods in Toxicology – domande e risposte

These are all Recostructed Human Epidermis that are useful to study chemicals and

mixtures but not formulations. The endpoint, analyzed through MTT test, is cytotoxicity.

+ CORROSITEX (not validated yet)

It’s an in vitro membrane barrier test. It has a limited application (it is useful only for certain acids,

bases and their derivates). It performs a colorimetric reaction.

13) Skin sensitization and Alternative Methods

Skin sensitization is an immune reaction to external substances in genetically predisposed subjects,

manifesting as itching, urticaria and eczema and it is possible to identify two different phases,

INDUCTION (= memory induction) and ELICITATION (= cell-mediated response) and 6 different steps

(key points):

1. Percutaneous absorption

2. Protein binding

3. Cytokine release

4. Activation of DC

5. Maturation and migration of DC

6. Antigen presentation to T lymphocytes helper

LLNA (TG 429) is one of the alternative methods. It means Local Lymph Nodes Assay and is an in vivo

model based on the “reduction-refinement method” compared to the Guinea Pig models. It provides

quantitative data; it reduces the number of animals used and their sufferance (no stimulation of skin

hypersensitive reaction and no use of adjuvants).

Sensitizers induce lymphocytes proliferation in lymph nodes evaluated through the Stimulation index

(SI), the ration between the proliferation of the lymphocytes in treated groups and that of the negative

controls.

The test uses female mice (nulliparous and not pregnant, aged between 8 and 12 weeks) with the

positive control being mercaptobenzothiazole.

Normally, the number of animals used is 20:

• Day 1-3 = application of the substance

• Day 6= injection of the 3H-methil thymidine (radioactive)

• After 5h = sacrifice of the animals —> isolation of the ear lymph nodes

• Cell suspension —> SI (measurement of incorporated 3H-methyl thiamidine)

SI > 2,5 = skin sensitizer

SI < 1,7 = no skin sensitizer

This test particularly assesses key point 6. However, following an AOP strategy, other key points can be

analyzed, such as:

- Key point 4 —> ARE-Nrf2 Luciferase test method (keratinocytes + luciferase)

5 Alternative Methods in Toxicology – domande e risposte

- Key point 2 —> DPRA (Direct Peptide Reactivity Assay)

It is an in chemico test that quantify the ability of a substance to bind a synthetic peptide

monitoring the cysteine and lysine depletion.

14) EDC definition

EDC stands for “Endocrine Disruptor chemicals”.

15) AOP definition

AOP stands for “Adverse Outcome Paghway”.

16) Endocrine Disruptor chemicals: testing approach

Endocrine disruptors are exogenous substances or mixtures that alter the function of the endocrine

system, causing adverse health effects in an organism, its progeny, or population. To study endocrine

disruptors, no single test can provide all the necessary information. Available tests target estrogen,

androgen, thyroid, or steroidogenesis (EATS) receptors. The testing strategy combines in vitro, in vivo,

and in silico methods.

It is possible to identify 5 different levels:

- Level 1 = all information (physico-chemical characteristics, standardized and non-

standardized test, epidemiological and filed studies and in silico studies)

- Level 2 = in vitro methods such as Estrogen or Androgen Receptor binding Affinity,

Steroidogenesis in vitro and thyroid disruption assay.

- Level 3 = in vivo studies to evaluate a specific mechanism

- Level 4 = in vivo studies to evaluate ad erse effects

- Level 5 = in vivo studies for more information

17) ITS: some examples

ITS stands for Integrated Testing Strategy.

Examples of ITS are:

- the AMES test + Micronucleus test

AMES Test: Also known as the Bacterial Reverse Mutation Test, it uses Salmonella

typhimurium mutant His-, which cannot biosynthesize histidine and requires exogenous histidine. To

determine if a substance is mutagenic, it is introduced into a culture of His- bacteria. After an

incubation period of 2 days at 37ºC, if histidine is present, it indicates that the substance caused the

bacteria to revert back, proving it is mutagenic.

Micronucleus Assay: This genotoxicity test detects micronuclei in the cytoplasm