Summary
Regulatory requirements
According to the current Notes for Guidance of the
Scientific Com... more Summary Regulatory requirements According to the current Notes for Guidance of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, for example, to ensure the light stability of cosmetics or used in sun protection products) should be tested for acute phototoxic and photogenotoxic potential. Testing for photosensitisation (immunological photoallergy) potential is not specifically required, but it is nevertheless often performed. Acute phototoxicity Due to a thorough multi-stage and multi-centre validation trial (1992–1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) had already gained acceptance by the SCCNFP in 1998, and it is recommended by the EMEA/CPMP as a basic preclinical test for acute phototoxicity. It was accepted as Method No. 41 in Annex V to Directive 67/548/EEC in the year 2000, and was accepted as the new Test Guideline 432 by the OECD in 2002. The 3T3-NRU-PT is regarded as a basic screen for identifying acute phototoxic potential. Two additional in vitro tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC-PT) and the Human 3-D Skin Model Phototoxicity Test (H3D-PT), are regarded as useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the RBC-PT permits an evaluation of the phototoxic mechanisms involved. In conclusion, the identification of acute phototoxic hazards is now regarded as being sufficiently covered by in vitro tests, so that animal testing for that endpoint can now be 100% replaced. Photogenotoxicity In the area of photogenotoxicity, almost the whole battery of in vitro genetic toxicity tests have been (or are currently being) converted into test protocols of photogenotoxicity tests. Tests exclusively predictive for gene mutation, for example, the Photo-Ames (P-Ames) Test and the Photo- Thymidine Kinase Test (P-TKT), have become less important than tests for clastogenic effects (for example, the Photo-Chromosome Aberration Test [P-CAT] and the Photo-Micronucleus Test [PMNT]. In addition, a number of promising indicator tests, such as the Photo-Comet Assay (P-Comet) have been developed. Although routinely used, to date none of the new photogenotoxicity tests have been formally validated. Therefore, the P-MNT and the P-Comet are currently being evaluated in a formal interlaboratory validation study. It is expected that these in vitro photogenotoxicity test methods may become available as validated and accepted methods within the next five years. Photoallergy (Photosensitisation) In the area of photoallergy (photosensitisation), as development of predictive in vitro tests for delayed contact sensitisation (allergenicity) potential without the involvement of light, due to a lack of ability to model the complex mechanisms underlying allergy, no promising in vitro methods to predict photo-sensitisation potential are currently in sight (see the section on skin sensitisation). One in vitro screening method, which models the covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while the binding of an excited chemical to proteins is a prerequisite for photoallergy, this is not a sufficient predictor on its own. The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (PLLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of “dark” sensitisation potential have been developed and accepted, their adaptation into similar photosensitisation testing will become possible.
About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in r... more About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which— after peer review and a long lasting international discussion—will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.
Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing ... more Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing of chemicals, the National Co-ordinators of OECD Test Guideline Programme endorsed in 2002 two new test guidelines: TG 430 'Transcutaneous Electrical Resistance assay' and TG 431 'Human Skin Model Test'.
It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cy... more It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cytotoxicity data to predict the in vivo starting doses, should be implemented into acute toxicity testing as soon as possible. The validity of the in vitro cytotoxicity data to establish appropriate starting doses for acute toxicity testing will be assessed experimentally. Secondly, in order to replace the use of animals in acute lethality testing a formal validation will be conducted in which the ability to predict rodent LD50 values and toxicity classes from cytotoxicity data will be evaluated.
The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employe... more The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employed for studying the fluorescence characteristics of tumor tissue in comparison with non tumorous tissue. Tumors were grown from the murine fibrosarcoma cell line SSK II and murine 3T3 fibroblasts (clone A31) were used for cultivating non tumorous tissue. Autofluorescence and xenofluorescence intensities induced by 5-aminolaevulinic
CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of em... more CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM
In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the E... more In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test...
This is the report of the thirty-fourth of a series of workshops organised by the European Centre... more This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best way...
ABSTRACT The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) wa... more ABSTRACT The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employed for studying the fluorescence characteristics of tumor tissue in comparison with non tumorous tissue. Tumors were grown from the murine fibrosarcoma cell line SSK II and murine 3T3 fibroblasts (clone A31) were used for cultivating non tumorous tissue. Autofluorescence and xenofluorescence intensities induced by 5-aminolaevulinic acid (5-ALA) were compared. Exogenous administration of 5-ALA, an early precursor in haem synthesis, induces accumulation of endogenous photoactive porphyrins, in particular protoporphyrin IX (PpIX). Fluorescence investigations were performed after 3-4d of incubation, when the tissues had reached macroscopically three dimensional stages of growth. Fluorescences were excited with a HBO-X 100 W lamp (Carl Zeiss) at a wavelength (lambda) equals 405 plus or minus 5 nm. Emissions were detected in the spectral range above 630 nm and visualized by real time digital image processing (Argus 10, HAMAMATSU) using an ICCD camera (HAMAMATSU). After administration of 0.4 mmolar 5-ALA solution to the CAM inoculated tissues the SSK II tumors exhibited higher fluorescence intensities than the 3T3 non tumorous tissues. Autofluorescence intensities of both types of tissues were not distinguishable. Furthermore, the effects of several biochemicals on the xenofluorescence intensities of the fibrosarcoma and fibroblast tissues were investigated.
European chemical policy in general and the REACH initiative in particular will increase the numb... more European chemical policy in general and the REACH initiative in particular will increase the number of chemical substances submitted to toxicological evaluation by several orders of magnitude compared to the current status. To limit animal exposure the resulting enormous increase in testing, however, asks for validated in vitro test systems. While the OECD favours in vitro testing for cutaneous absorption using viable human and animal skin (Guideline 428) the availability of viable human skin is already limited today.
Summary
Regulatory requirements
According to the current Notes for Guidance of the
Scientific Com... more Summary Regulatory requirements According to the current Notes for Guidance of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, for example, to ensure the light stability of cosmetics or used in sun protection products) should be tested for acute phototoxic and photogenotoxic potential. Testing for photosensitisation (immunological photoallergy) potential is not specifically required, but it is nevertheless often performed. Acute phototoxicity Due to a thorough multi-stage and multi-centre validation trial (1992–1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) had already gained acceptance by the SCCNFP in 1998, and it is recommended by the EMEA/CPMP as a basic preclinical test for acute phototoxicity. It was accepted as Method No. 41 in Annex V to Directive 67/548/EEC in the year 2000, and was accepted as the new Test Guideline 432 by the OECD in 2002. The 3T3-NRU-PT is regarded as a basic screen for identifying acute phototoxic potential. Two additional in vitro tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC-PT) and the Human 3-D Skin Model Phototoxicity Test (H3D-PT), are regarded as useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the RBC-PT permits an evaluation of the phototoxic mechanisms involved. In conclusion, the identification of acute phototoxic hazards is now regarded as being sufficiently covered by in vitro tests, so that animal testing for that endpoint can now be 100% replaced. Photogenotoxicity In the area of photogenotoxicity, almost the whole battery of in vitro genetic toxicity tests have been (or are currently being) converted into test protocols of photogenotoxicity tests. Tests exclusively predictive for gene mutation, for example, the Photo-Ames (P-Ames) Test and the Photo- Thymidine Kinase Test (P-TKT), have become less important than tests for clastogenic effects (for example, the Photo-Chromosome Aberration Test [P-CAT] and the Photo-Micronucleus Test [PMNT]. In addition, a number of promising indicator tests, such as the Photo-Comet Assay (P-Comet) have been developed. Although routinely used, to date none of the new photogenotoxicity tests have been formally validated. Therefore, the P-MNT and the P-Comet are currently being evaluated in a formal interlaboratory validation study. It is expected that these in vitro photogenotoxicity test methods may become available as validated and accepted methods within the next five years. Photoallergy (Photosensitisation) In the area of photoallergy (photosensitisation), as development of predictive in vitro tests for delayed contact sensitisation (allergenicity) potential without the involvement of light, due to a lack of ability to model the complex mechanisms underlying allergy, no promising in vitro methods to predict photo-sensitisation potential are currently in sight (see the section on skin sensitisation). One in vitro screening method, which models the covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while the binding of an excited chemical to proteins is a prerequisite for photoallergy, this is not a sufficient predictor on its own. The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (PLLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of “dark” sensitisation potential have been developed and accepted, their adaptation into similar photosensitisation testing will become possible.
About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in r... more About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which— after peer review and a long lasting international discussion—will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.
Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing ... more Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing of chemicals, the National Co-ordinators of OECD Test Guideline Programme endorsed in 2002 two new test guidelines: TG 430 'Transcutaneous Electrical Resistance assay' and TG 431 'Human Skin Model Test'.
It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cy... more It was suggested in the ICCVAM workshop that the Register of Cytotoxicity (RC), using in vitro cytotoxicity data to predict the in vivo starting doses, should be implemented into acute toxicity testing as soon as possible. The validity of the in vitro cytotoxicity data to establish appropriate starting doses for acute toxicity testing will be assessed experimentally. Secondly, in order to replace the use of animals in acute lethality testing a formal validation will be conducted in which the ability to predict rodent LD50 values and toxicity classes from cytotoxicity data will be evaluated.
The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employe... more The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employed for studying the fluorescence characteristics of tumor tissue in comparison with non tumorous tissue. Tumors were grown from the murine fibrosarcoma cell line SSK II and murine 3T3 fibroblasts (clone A31) were used for cultivating non tumorous tissue. Autofluorescence and xenofluorescence intensities induced by 5-aminolaevulinic
CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of em... more CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM
In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the E... more In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test...
This is the report of the thirty-fourth of a series of workshops organised by the European Centre... more This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best way...
ABSTRACT The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) wa... more ABSTRACT The in vivo model of the chorioallantoic membrane of fertilized chicken embryos (CAM) was employed for studying the fluorescence characteristics of tumor tissue in comparison with non tumorous tissue. Tumors were grown from the murine fibrosarcoma cell line SSK II and murine 3T3 fibroblasts (clone A31) were used for cultivating non tumorous tissue. Autofluorescence and xenofluorescence intensities induced by 5-aminolaevulinic acid (5-ALA) were compared. Exogenous administration of 5-ALA, an early precursor in haem synthesis, induces accumulation of endogenous photoactive porphyrins, in particular protoporphyrin IX (PpIX). Fluorescence investigations were performed after 3-4d of incubation, when the tissues had reached macroscopically three dimensional stages of growth. Fluorescences were excited with a HBO-X 100 W lamp (Carl Zeiss) at a wavelength (lambda) equals 405 plus or minus 5 nm. Emissions were detected in the spectral range above 630 nm and visualized by real time digital image processing (Argus 10, HAMAMATSU) using an ICCD camera (HAMAMATSU). After administration of 0.4 mmolar 5-ALA solution to the CAM inoculated tissues the SSK II tumors exhibited higher fluorescence intensities than the 3T3 non tumorous tissues. Autofluorescence intensities of both types of tissues were not distinguishable. Furthermore, the effects of several biochemicals on the xenofluorescence intensities of the fibrosarcoma and fibroblast tissues were investigated.
European chemical policy in general and the REACH initiative in particular will increase the numb... more European chemical policy in general and the REACH initiative in particular will increase the number of chemical substances submitted to toxicological evaluation by several orders of magnitude compared to the current status. To limit animal exposure the resulting enormous increase in testing, however, asks for validated in vitro test systems. While the OECD favours in vitro testing for cutaneous absorption using viable human and animal skin (Guideline 428) the availability of viable human skin is already limited today.
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Papers by M. Liebsch
Regulatory requirements
According to the current Notes for Guidance of the
Scientific Committee on Cosmetic Products and
Non-Food Products (SCCNFP), cosmetic ingredients
and mixtures of ingredients absorbing UV
light (in particular UV filter chemicals used, for
example, to ensure the light stability of cosmetics or
used in sun protection products) should be tested
for acute phototoxic and photogenotoxic potential.
Testing for photosensitisation (immunological photoallergy)
potential is not specifically required, but
it is nevertheless often performed.
Acute phototoxicity
Due to a thorough multi-stage and multi-centre validation
trial (1992–1998) the In Vitro 3T3 Neutral
Red Uptake Phototoxicity Test (3T3-NRU-PT) had
already gained acceptance by the SCCNFP in 1998,
and it is recommended by the EMEA/CPMP as a
basic preclinical test for acute phototoxicity. It was
accepted as Method No. 41 in Annex V to Directive
67/548/EEC in the year 2000, and was accepted as
the new Test Guideline 432 by the OECD in 2002.
The 3T3-NRU-PT is regarded as a basic screen for
identifying acute phototoxic potential.
Two additional in vitro tests, formally evaluated
in controlled blind trials, the RBC Phototoxicity
Test (RBC-PT) and the Human 3-D Skin Model
Phototoxicity Test (H3D-PT), are regarded as useful
and important adjunct tests to overcome some
limitations of the 3T3-NRU-PT, namely the fairly
low UVB tolerance of the 3T3 fibroblasts and the
inability to model the bioavailability of test materials
topically applied to the skin. In addition, the
RBC-PT permits an evaluation of the phototoxic
mechanisms involved. In conclusion, the identification
of acute phototoxic hazards is now regarded as
being sufficiently covered by in vitro tests, so that
animal testing for that endpoint can now be 100%
replaced.
Photogenotoxicity
In the area of photogenotoxicity, almost the whole
battery of in vitro genetic toxicity tests have been
(or are currently being) converted into test protocols
of photogenotoxicity tests. Tests exclusively
predictive for gene mutation, for example, the
Photo-Ames (P-Ames) Test and the Photo-
Thymidine Kinase Test (P-TKT), have become less
important than tests for clastogenic effects (for
example, the Photo-Chromosome Aberration Test
[P-CAT] and the Photo-Micronucleus Test [PMNT].
In addition, a number of promising indicator
tests, such as the Photo-Comet Assay (P-Comet)
have been developed. Although routinely used, to
date none of the new photogenotoxicity tests have
been formally validated. Therefore, the P-MNT and
the P-Comet are currently being evaluated in a formal
interlaboratory validation study. It is expected
that these in vitro photogenotoxicity test methods
may become available as validated and accepted
methods within the next five years.
Photoallergy (Photosensitisation)
In the area of photoallergy (photosensitisation), as
development of predictive in vitro tests for delayed
contact sensitisation (allergenicity) potential without
the involvement of light, due to a lack of ability
to model the complex mechanisms underlying
allergy, no promising in vitro methods to predict
photo-sensitisation potential are currently in sight
(see the section on skin sensitisation). One in vitro
screening method, which models the covalent binding
of a light activated chemical to human serum
albumin, may become relevant. However, while the
binding of an excited chemical to proteins is a prerequisite
for photoallergy, this is not a sufficient
predictor on its own. The only promising alternatives
currently under development are in vivo
refinements, like the Photo Local Lymph Node
Assay (PLLNA). Once a reliable and predictive in
vitro test battery and strategy for the assessment of
“dark” sensitisation potential have been developed
and accepted, their adaptation into similar photosensitisation
testing will become possible.
Regulatory requirements
According to the current Notes for Guidance of the
Scientific Committee on Cosmetic Products and
Non-Food Products (SCCNFP), cosmetic ingredients
and mixtures of ingredients absorbing UV
light (in particular UV filter chemicals used, for
example, to ensure the light stability of cosmetics or
used in sun protection products) should be tested
for acute phototoxic and photogenotoxic potential.
Testing for photosensitisation (immunological photoallergy)
potential is not specifically required, but
it is nevertheless often performed.
Acute phototoxicity
Due to a thorough multi-stage and multi-centre validation
trial (1992–1998) the In Vitro 3T3 Neutral
Red Uptake Phototoxicity Test (3T3-NRU-PT) had
already gained acceptance by the SCCNFP in 1998,
and it is recommended by the EMEA/CPMP as a
basic preclinical test for acute phototoxicity. It was
accepted as Method No. 41 in Annex V to Directive
67/548/EEC in the year 2000, and was accepted as
the new Test Guideline 432 by the OECD in 2002.
The 3T3-NRU-PT is regarded as a basic screen for
identifying acute phototoxic potential.
Two additional in vitro tests, formally evaluated
in controlled blind trials, the RBC Phototoxicity
Test (RBC-PT) and the Human 3-D Skin Model
Phototoxicity Test (H3D-PT), are regarded as useful
and important adjunct tests to overcome some
limitations of the 3T3-NRU-PT, namely the fairly
low UVB tolerance of the 3T3 fibroblasts and the
inability to model the bioavailability of test materials
topically applied to the skin. In addition, the
RBC-PT permits an evaluation of the phototoxic
mechanisms involved. In conclusion, the identification
of acute phototoxic hazards is now regarded as
being sufficiently covered by in vitro tests, so that
animal testing for that endpoint can now be 100%
replaced.
Photogenotoxicity
In the area of photogenotoxicity, almost the whole
battery of in vitro genetic toxicity tests have been
(or are currently being) converted into test protocols
of photogenotoxicity tests. Tests exclusively
predictive for gene mutation, for example, the
Photo-Ames (P-Ames) Test and the Photo-
Thymidine Kinase Test (P-TKT), have become less
important than tests for clastogenic effects (for
example, the Photo-Chromosome Aberration Test
[P-CAT] and the Photo-Micronucleus Test [PMNT].
In addition, a number of promising indicator
tests, such as the Photo-Comet Assay (P-Comet)
have been developed. Although routinely used, to
date none of the new photogenotoxicity tests have
been formally validated. Therefore, the P-MNT and
the P-Comet are currently being evaluated in a formal
interlaboratory validation study. It is expected
that these in vitro photogenotoxicity test methods
may become available as validated and accepted
methods within the next five years.
Photoallergy (Photosensitisation)
In the area of photoallergy (photosensitisation), as
development of predictive in vitro tests for delayed
contact sensitisation (allergenicity) potential without
the involvement of light, due to a lack of ability
to model the complex mechanisms underlying
allergy, no promising in vitro methods to predict
photo-sensitisation potential are currently in sight
(see the section on skin sensitisation). One in vitro
screening method, which models the covalent binding
of a light activated chemical to human serum
albumin, may become relevant. However, while the
binding of an excited chemical to proteins is a prerequisite
for photoallergy, this is not a sufficient
predictor on its own. The only promising alternatives
currently under development are in vivo
refinements, like the Photo Local Lymph Node
Assay (PLLNA). Once a reliable and predictive in
vitro test battery and strategy for the assessment of
“dark” sensitisation potential have been developed
and accepted, their adaptation into similar photosensitisation
testing will become possible.