CREDIT SEMINAR-IICREDIT SEMINAR-II

Major Advisor : Dr. K. JayakumarSpeaker : Amol R. Padol

80,000 chemicals - currently in use2000 new chemicals - introduced annually (OTA , 1995)

The public and the environment come in contact with these substances during their manufacture, distribution, use and disposal.

To safe guard humanity and their surroundings, toxicological tests are required for which guidelines are issued by regulatory agencies.

The information generated from these test methods is used for pre-market evaluation, hazard classification and risk assessment.

The potential adverse effects of chemicals are currently assessed largely by tests involving laboratory animals.

The scientific community influenced by,

advances in the understanding of molecular and cellular mechanisms of toxicity.

Desire to develop tests that will be more predictive of potential chemical toxicity.

There is also great interest to develop tests that are more cost and time efficient (Stokes & Marafante, 1998).

In Vitro studies Studies which do not use multicellular whole organisms, but

rather microorganisms or material isolated from whole organisms, or simulations thereof as test systems.

In-vitro versus In-vivo provides potential to be more rigorously standardized than

in vivo tests. more reliable since quality data can be generated less expensive human cells can be used directly as test systems offer good experimental control of the cellular doses of

chemicals

the production of “reversible damage of the skin following the application of a test substance for up to 4 hours” (OECD TG 404, 2002).

the potential of a certain substance to cause erythema or eschar or oedema after a single topical application.

Dermal irritation

Eye irritation

The production of changes in the eye following the application of a test substance to the anterior surface of the eye, which are fully reversible within 21 days of application (OECD TG 405, 2002).

In vivo irritation studies for the eye and skin assess the short term effects of materials (Gad, 1997).

These tests have been in existence since the 1930's.

These in vivo methods used rabbits for evaluation of materials for their potential to cause dermal and eye irritation.

Historical Background Marshall Hall was one of the first to address the issue of

alternatives- 1876.

In 1969, the Fund for the Replacement of Animals in Medical Experimentation (FRAME) was founded in the UK.

1981- establishment of the John Hopkins Centre for Alternatives to Animal Testing (CAAT).

The initial efforts of CAAT focused on the establishment of a firm scientific foundation of alternative testing methodologies.

1990- The European Centre for the Validation of Alternative Methods (ECVAM) was established.

During 1999-2001, some of the most promising in vitro methods were evaluated in prevalidation studies.

ECVAM started skin irritation validation study (SIVS) in Nov. 2003.

In April 2007, ECVAM approved 2 alternative test, EpiSkin and EpiDerm Skin Irritation Tests as replacements of the in vivo rabbit skin irritation test.

2008- devlopment of SkinEthic, Human Oral Epithelium model

Validation …? Validation is the process by which the reliability and

relevance of an alternative method are established for a particular purpose (Balls et al., 1990).

Reliability - establishing the reproducibility of toxicity hazard predictions within and between laboratories and overtime (Bruner et al., 1996).

Relevance - establishing the scientific usefulness of

results from an alternative method (Frazier, 1990).

If an alternative method is judged both reliable and relevant at the end of the validation process, then the new assay should be considered validated.

Once validated the alternative method may be used routinely in safety assessment process and may be considered for acceptance by regulatory agencies (Zeiger and Stokes, 1998).

Conventional Method- eye irritationOECD TG 405 (2002)OPPTS 870.2400 (EPA, August 1998)

Test system- albino rabbit The substance to be tested is applied in

a single dose to one of the eyes of the experimental animal; the untreated eye serves as the control.

The eyes of the test animals washed after 24 h following instillation of the test substance.

Dose level: Liquids - 0.1 ml solids, pastes, and particulate substances - volume of 0.1 ml

or a weight not more than 100 mg. Aerosols- test substance administered to the eye in a burst

of about one second, from a distance of 10 cm.

Observations of eye reactions:examined at 1, 24, 48 and 72 h after application.

Cornea- OpacityIris- congestion, swelling, circumcorneal hyperaemia,

hemorrhages.Conjunctiva- Redness and hyperaema.

OECD TG 404 (2002)OPPTS 870.2500 (EPA, August 1998) Test system- albino rabbit The substance to be tested is applied in a single dose to the

skin of animal; untreated skin areas of the test animal serve as the control.

Dose level: liquid- 0.5 mlsolid or paste- 0.5 g

exposure period – 4 h

Conventional Method- skin irritation

Application of the test substanceThe test substance should be applied to a small area (6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape.

Observations of skin reactionsScored at 60 minutes, and then at 24, 48 and 72 h after patch removal.

Examined for signs of erythema and oedema

Deficiencies of the Draize Skin and Eye Tests

These are unable to obtain important information concerning mechanisms of toxicity of test chemicals.

Inadequate objectivity in obtaining irritancy scores.

expenditures related to the large numbers of animals required and time-consuming evaluation.

Irreproducibility of results

Issue of variability within the test (Davila et al., 1998).

Anatomical considerations

Presence of a nictitating membrane

Larger conjunctival sac

Thinner cornea

Less tear production……………………..the rabbit is generally considered on

overly sensitive model for humans.

Organotypic models or tissue and cell culture systems.

At present there are two OECD TG based on the use of in vitro eye irritation tests.

1. Bovine Corneal Opacity and Permeability (BCOP) Test (OECD TG 437) - 7 Sept 2009

2. Isolated Chicken Eye (ICE) Test, (OECD TG 438) -7 Sept 2009

Alternative Assays for Ocular Irritation 

Neutral Red (NR) release assay (Reader et al., 1990) NR uptake assay (Jones et al., 1999) Tissue equivalent assay (TEA) (Southee et al., 1999) Chinese hamster lung (CHL) cell lines Bovine corneal opacity and permeability (BCOP) assay Isolated chicken eye (ICE) test Hens egg test on the chorio-allontoic membrane (HET-

CAM)

Alternative Assays for Ocular Irritation 

Bovine corneal opacity and permeability (BCOP) assay (Gautheron et al., 1992)

First scientifically valid alternative methods to gain regulatory acceptance for ocular safety testing.

OECD TG 437 (2009)

Not a complete replacement for the rabbit eye test

Recommended for use as part of a tiered testing strategy for regulatory classification and labeling.

ProtocolEyes are collected from slaughter house

immersed in Hanks’ Balanced Salt Solution

corneas free of defects are dissected

mounted in specially designed corneal holders Eagle's Minimum Essential Medium (EMEM)

32 ± 1°C for 1 hour

test article is applied as a single dose for 10 min

Post exposure period- 4 h

Endpoints MeasuredOpacity- opacitometer Permeability - determined by the amount of sodium

fluorescein dye penetration.

Limitations false positive for alcohols and ketones does not consider conjunctival and iridal injuries does not allow for an assessment of systemic toxicity

associated with ocular exposure

Isolated Chicken Eye (ICE) Test

Gained regulatory acceptance for ocular safety testing (OECD TG 438, 2009).

Not a complete replacement for the rabbit eye test

Recommended for use as part of a tiered testing strategy for regulatory classification and labeling.

ProtocolEyes isolated from chickens (slaughter house)

placed in a superfusion apparatus(isotonic saline)

test article is applied as a single dose for 10 sec(Liquids 0.03 mLSolids 0.03 g)

Corneal reactions are measured up to 4 h post-treatmentFluorescein retention is evaluated at 30 min post-treatment

End points- Corneal swelling, opacity and fluorescein retention

Limitations: false positive for alcohols does not consider conjunctival and iridal injuries does not allow for assessment of systemic toxicity

associated with ocular exposure

Hens egg test on the chorio-allontoic membrane (HET-CAM)

The CAM of the developing chicken egg is considered to be a suitable model to study irritation of mucous tissues (Spielmann et al., 1997).

The CAM of an embryonated hen’s egg is similar to the vascularized mucosal tissues of the eye.

9 day fertilized hen’s eggs

The CAM (25%) is treated with the test item for 30 sec

test item is washed off

effects are assessed within 5 minutes

Endpoint – Coagulation, hyperaemia and haemorrhageThe time for each reaction recorded in sec to calculate

irritation score

Solids- 0.3gLiquids- 0.3 ml

Human Tissue Models- EpiOcular

EpiOcular is currently under validation as a replacement to Draize test by ECVAM (ECVAM, 2010)

Water insoluble and soluble materials

End point – cytotoxicity

Human Reconstituted Corneal Epithelium

MatTek Corp, USA

Alternative Assays for Skin Irritation

Number of in vitro alternatives have been proposed but there are no in vitro tests for replacing the classical Draize test.

Promising in vitro methods : EpiDerm™, EpiSkin™, the non-perfused pig ear model.

EpiDerm and EpiSkin are validated for the purpose of classification and labelling.

Reconstructed Human Epidermis (RhE) Test Method

As a partial replacement test, within a tiered testing strategy.

It covers the initial step of the inflammatory cascade (cell damage resulting in localised trauma) that occurs during irritation in vivo.

Applicable to solids, liquids, semi-solids and waxes.

Test system

Non-transformed human-derived epidermal keratinocytes.

Cultured to form a multilayered, highly differentiated model of human epidermis

Consists of organized basal, spinous and granular layers, and a multilayered stratum corneum analogous to those found in vivo.

Reconstituted Human Epidermis Model

dose :Liquid test article- 25 μL/cm2

Solid test article- 25 mg/cm2

exposure period - varies between 15 and 60 min incubation temperature - between 20 and 37°C controls :Positive control- 5% aqueous SDS Negative control- water or phosphate buffered saline (PBS).

MTT - cell viability assay

Reduction of yellow 3-(4,5-dimethythiazol)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase.

MTT reduced to an insoluble, dark purple formazan.

The cells are then solubilised with an organic solvent (eg. isopropanol) and the released, formazan reagent is measured spectrophotometrically.

Reduction of MTT only occur in metabolically active cells. The level of activity is a measure of the viability of the cells (Mosmann, 1983)

Reconstituted Human Epidermis Models (RHE) EpiSkinTM model (L’Oreal, France)

EpiDermTM model (MatTek Corp., USA)

SkinEthicTM model (Skinethic, Nice, France)

EpiSkin

Developed by L’Oreal, France and commercially supplied by SkinEthic Laboratories, France.

ECVAM validated and recognized as a stand alone method for screening and replacement (ECVAM, 2010)

It has a reconstructed epidermis with a functional stratum corneum.

Endpoint – Cell viability, Interleukin -1 α release.

Exposure period- 15 min. Post exposure- 42 h

cytotoxicity measurement using MTT reduction and Interleukin -1 α assay (Fentem et al., 1998).

Assessment of irritation:cell viability ≤ 50% after 15 min exposure Interleukin -1 α release > 60 pg/mL

Protocol

EpiDerm (Kubilus et al., 1996)

It incorporates normal human keratinocytes cultured

on permeable millipore membranes.

Endpoint – The time taken to reduce cell viability by

50%.

The EpiDerm test do not qualify as a stand-alone replacement but is recommended for the identification of irritant chemicals (ECVAM,2009).

Predictive capacity of in vitro human skin models

Assay Sensitivity Specificity

Episkin (MTT endpoint) 75% 81%

Episkin (MTT + IL-1α endpoints) 91% 79%

EpiDerm 57% 84%

Sensitivity - % in vivo irritants correctly identified by assay

Specificity - % in vivo non-irritants correctly identified by assay.

(ECVAM, 2007)

A great deal of effort has been directed toward development and validation of alternative methods for ocular and dermal irritation testing.

Some alternative tests for reduction and refinement have been validated and accepted by international authorities (OECD) and can be used for safety assessment.

Compared to the Draize test the in vitro model based on human cells is expected to predicts the better range of responses to toxic injury that occur in the human eye and skin.

Conclusions