In vitro alternatives for skin and eye irritation studies

1. CREDIT SEMINAR-IICREDIT SEMINAR-II
Major Advisor : Dr. K. Jayakumar
Speaker : Amol R. Padol

2.  80,000 chemicals - currently in use
2000 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.

3.  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.

4. 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).

5. 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

6.  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

7. 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).

8.  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.

9. 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.

10.  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

11. 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).

12.  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).

13. Conventional Method- eye irritation
OECD 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.

14. 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- Opacity
Iris- congestion, swelling, circumcorneal hyperaemia,
hemorrhages.
Conjunctiva- Redness and hyperaema.

15. 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 ml
solid or paste- 0.5 g
 exposure period – 4 h
Conventional Method- skin irritation

16.  Application of the test substance
The 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 reactions
Scored at 60 minutes, and then at 24, 48 and 72 h
after patch removal.
Examined for signs of erythema and oedema

17. 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).

18. 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.

19.  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

20.  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

21. 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.

22. Protocol
Eyes 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

23. test article is applied as a single dose for 10 min
Post exposure period- 4 h
Endpoints Measured
Opacity- opacitometer
Permeability - determined by the amount of sodium
fluorescein dye penetration.

24. 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

25. 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.

26. Protocol
Eyes 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 mL
Solids 0.03 g)
Corneal reactions are measured up to 4 h post-treatment
Fluorescein retention is evaluated at 30 min post-treatment

27.  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

28. 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.

29. 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 haemorrhage
The time for each reaction recorded in sec to calculate
irritation score
Solids- 0.3g
Liquids- 0.3 ml

30. 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

31. 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.

32. 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.

33. 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

34.  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).

35. 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)

36. Reconstituted Human Epidermis Models
(RHE)
 EpiSkinTM
model (L’Oreal, France)
 EpiDermTM
model (MatTek Corp., USA)
 SkinEthicTM
model (Skinethic, Nice, France)

37. 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.

38.  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

39. 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).

40. 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)

41.  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