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Directorate-General for Health and Food Safety
Opinion on the use of alternative methods to animal testing in the safety evaluation of cosmetic ingredients or mixtures of ingredients (with three annexes) and the updating of notes of guidance for testing of cosmetic ingredients for their safety evaluation (with two annexes) adopted by the SCCNFP at the plenary meeting of 20 January 1999
a- the status of alternative methods to animal testing for the safety assessment of cosmetic ingredients in January 1999. The SCCNFP is of the opinion that three in vitro methods to assess skin corrosivity and phototoxicity have been validated and can be considered applicable to the safety evaluation of cosmetic ingredients or mixtures of ingredients. These methods are the "Rat Skin Transcutaneous Electrical Resistance Test" and the "EPISKIN Test" for the assessment of skin corrosivity, and the "3T3 Neutral Red Uptake Phototoxicity Test" for the assessment of phototoxicity.
The SCCNFP has considered the possible usefulness of the various existing in vitro methodologies to evaluate the percutaneous absorption of cosmetic ingredients and will define a set of minimal criteria needed for the acceptance of in vitro percutaneous absorption studies for the evaluation of toxicological profile of cosmetic ingredients.
b- the updating of the Notes of Guidance (XXIV/1878/97) which includes guidelines on the use of human volunteers in the testing of potentially cutaneous irritant cosmetic ingredients or mixtures of ingredients and an opinion on the microbiological quality of finished cosmetic products.
c- the issues regarding alternative methods and the dossier to be discussed during the year 1999.
1.Mandate to the SCCNFP (XXIV/1890/98)
1.1 The SCCNFP shall act as a resource of scientific expertise to the European Commission, with regards to the development of alternative methods. To that effect they will regularly meet with representatives from the concerned parties to :
However, such advice in no way prejudices the right of the SCCNFP to reject data previously discussed, if the scientific standard of the work is considered inappropriate.
Although this mandate has been developed in relation to cosmetic ingredients, it is envisaged that scientifically sound non-animal methods for safety assessment will have broad application.
1.2. The SCCNFP was requested to continuously update the Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation as stated on page 8 of the 2 nd Revision, adopted by the former SCC (Scientific Committee on Cosmetology) on January 16 th, 1997.
Due to the almost complete review of the former SCC, and the presence in the SCCNFP of experts on other disciplines, it was decided to include technical modifications to the content of the Notes, such as Guidelines for Human Testing, as it seems appropriate to give advice on this new aspects.
2. Background
In a note from the responsible Commission Service of August 11 th 1998, with the subject to review on the status of alternative methods to animal testing in the safety evaluation of cosmetic ingredients, it is stated:
"... it would be appreciated if the summary on the status of alternative methods for safety assessment of cosmetic ingredients could be available by the end of January 1999 at the latest".
The Specific Working Party (SPW) of the SCCNFP "Alternatives to Animal Testing & Dossiers" was established on November 14 th 1997 (during the 1 st Plenary Meeting of the SCCNFP) and was put into operation in January 1998. The SPW met on Feb. 16 th, March 4-5 th, March 24-25 th, April 20-21 st, May 12-13 th, June 16 th, Sept. 22 nd, Nov. 24 th, Dec. 10 th. It met with representatives of the concerned Commission Services, of ECVAM (European Center for the Validation of Alternative Methods) and with representatives of the European Cosmetic Industry.
3.Opinion on the Use of Alternative Methods to Animal Testing in the Safety Evaluation of Cosmetic Ingredients or Mixture of Ingredients
3.1. In Vitro Methods to Assess Skin Corrosivity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients
ECVAM has concluded positively the validation of two in vitro methods to assess the skin corrosivity potential of several chemicals, including 60 agents employed as cosmetic ingredients.
The SCCNFP considers that in the safety evaluation of chemicals intended to be used as cosmetic ingredients, when corrosivity potential cannot be excluded, they should be tested by the "Rat Skin Transcutaneous Electrical Resistance (TER) Test" or by the "EPISKIN Test", before testing for irritancy on animals or humans. An opinion of the SCCNFP has been adopted during its Plenary Session on November 25 th, 1998 (SCCNFP/0070/98Final, November 1998).
The guideline on the use of TER and EPISKIN methods has been presented by ECVAM to OECD (98/III/COS/21) and it is now ready for approval. Annex 1.
3.2. In Vitro Methods to Assess Phototoxicity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients
As reported in the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97) adopted by the former SCC, all chemicals which can absorb UVA and/or UVB light may change their molecular configuration and may undergo further biological reactions of toxicological relevance for consumers. The former SCC as well as the present SCCNFP request for all such chemicals routine testing for phototoxicity.
Animal models have not been validated for testing for phototoxicity.
ECVAM, in cooperation with COLIPA (European Cosmetic, Toilettry and Perfumery Association), has concluded a series of studies representing pre-validation, validation and application to cosmetic ingredients of one in vitro method, named "3T3 Neutral Red Uptake, Phototoxicity Test (3T3 NRU PT)".
The principle of the method is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of UVA/visible light.
The predictive value of this method for a potential human phototoxic chemical has been demonstrated to be between 95 and 100%.
The method has been demonstrated to be able to identify UV filter chemicals which are non-phototoxic for consumers and which are employed in cosmetic products. A guideline for testing UV light absorbing chemicals by using 3T3 NRU PT has been presented to OECD.
An opinion of the SCCNFP has been adopted during its Plenary Session on November 25 th, 1998, proposing the use of 3T3 NRU PT test as a method for testing UV light absorbing cosmetic ingredients, or mixtures of ingredients for phototoxic potential (SCCNFP/0069/98 Final, November 1998): Annex 2.
3.3. In Vitro Methods to Assess Percutaneous Absorption of Cosmetic Ingredients
The assessment of the percutaneous absorption of a given cosmetic ingredient has primary relevance in the procedure for evaluating the safety of cosmetics for consumers. The need to develop a series of toxicological studies as a new cosmetic ingredient depends very much on the knowledge of the percutaneously absorbed dose which could be distributed systemically in the human body (XXIV/1878/97).
In the last few years, two OECD Proposals to evaluate percutaneous absorption by in vivo or in vitro methods have been presented, but none of them has been accepted in the regulatory system yet.
COLIPA has presented a technical document on in vitro/ in vivo percutaneous absorption, reporting data and protocols in use since many years by several cosmetic companies, in the evaluation of the safety of hair dyes, UV filters and some other cosmetic ingredients.
The data presented include technical information on methodologies used and contain technical details in relation to the systems developed to assess percutaneous absorption, the application of the test substance and the conditions during the analysis, the type of the skin and the species from which it derives, a series of comparisons between different species of skin and different systems (namely in vitro Vs in vivo) some intra-assay and inter-laboratory comparisons, the evaluation of the results.
The SCCNFP has reviewed this documentation submitted by COLIPA and agrees with the rationale for using in vitro methods to evaluate the percutaneous absorption as it was already recommended in the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97).
However, the SCCNFP considers relevant to define in a short time a set of minimal criteria needed for the acceptance of in vitro percutaneous absorption studies to be evaluated when the dossiers of these cosmetic ingredients for their inclusion in the Annexes of Cosmetic Directive 76/768/EEC are submitted.
An opinion of the SCCNFP was adopted during its Plenary Meeting on January 20 th 1999, proposing the use of in vitro methods to assess percutaneous absorption of cosmetic ingredients (SCCNFP/0088/98 Rev 2, January 1999): Annex 3.
4. Updating of Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation
4.1. Guidelines on the Use of Human Volunteers in the Testing of Potentially Cutaneous Irritant Cosmetic Ingredients or Mixtures of Ingredients
In the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97) the former SCC stated that "for an analysis of potential adverse effects of a cosmetic product or ingredient (e.g. irritation, sensitization, non-invasive cutaneous penetration studies) observations on human subjects should be used if available". This opinion was recently expressed also by the present SCCNFP, by considering that tests on animals for skin irritation or (not yet) validated alternative methods may be limited regarding their predictive value for exposure of a human population. The SCCNFP states that confirmatory tests on humans should be scientifically and ethically necessary, provided that the toxicological profile of an ingredient or a mixture of ingredients based on animal or alternative methods is available and that a high degree of safety is to be expected (SCCNFP/0003/98). Therefore, an opinion on the use of human volunteers in the testing of potentially cutaneous irritant cosmetic ingredients has been adopted by the SCCNFP in its plenary session of November 25 th, 1998 (SCCNFP/0003/98 Final, November 1998): Annex 4.
This document presents the general consensus of the SCCNFP regarding one aspect of human testing, however stating again that, at present, human testing of cosmetic ingredients should not be preferred to animal testing and that the safety testing of cosmetics on humans may not be considered to be an alternative method to the use of animals.
The present opinion also stresses the concept that confirmatory skin tolerance tests of cosmetics in humans are subject to ethical concern. According to the Declaration of Helsinki "safety studies of cosmetics in human must be subjected to National Regulations and Good Clinical Practice for Trials on Medicinal Products in the European Union".
Test protocols to be applied in the safety evaluation of cosmetic ingredients or their mixtures with human studies have not been validated yet. SCCNFP's opinion is that a set of specific guidelines concerning the most applied protocols can be prepared in the near future and presented to the European Commission. The SCNFP has been requested by the responsible Commission Service to offer guidance on:
A draft proposal of "Guidelines on the Testing of Cosmetic Finished Products on Human Volunteers" is currently under preparation and is being discussed by the SCCNFP. An opinion should soon be available.
4.2. Revision of Annex 7 of Notes of Guidance. Microbiological Quality of Finished Cosmetic Products (XXIV/1878/97)
As stated several times by the former SCC, the "Notes of Guidance..." will require future amendments as scientific knowledge advances. A second reason for revising the document will depend on the improvement of cosmetic technologies in the industrial sector.
On September 23 rd 1998 the SCCNFP has adopted the Revision of Annex 7. "Microbiological Quality of the Finished Products", as amendment to the previous document (SCCNFP/0004/98 Final). Annex 5.
5. Work Programme for 1999
The work planned for 1999 in fulfillment of the Commission's request is schematically represented by the following list:
ANNEX 1
SCCNFP/0070/98 Final - November 1998
The Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers
OPINION ON IN VITRO METHODS TO ASSESS SKIN CORROSIVITY IN THE SAFETY EVALUATION OF COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
Terms of Reference
Two in vitro methods developed to assess skin corrosivity of chemicals, the "Rat skin Trancutaneous Electrical Resistance (TER)test" and the "EPISKIN test" have been validated by ESAC (ECVAM Scientific Advisory Committee).
The Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) has been requested by DG III to advise the Commission on the applicability of the methods to the safety assessment of chemicals used as cosmetic ingredients.
1- Background
The European Centre for the Validation of Alternative Methods (ECVAM) has conducted in 1996-1997 a validation study of in vitro tests developed to assess skin corrosivity of chemicals. This study was a follow-up to a pre-validation study of tests developed for replacing the in vivo Draize skin corrosivity test in rabbits.
The main objectives of the validation study, as defined by the sponsors and the management team before the study began, were :
(a) to identify tests capable of discriminating corrosives (C) from non corrosive (NC) for selected groups of chemicals (e.g. organic acids, phenols) and/or all chemicals (single chemical entities only);
(b) to determine whether the tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals.
2- Organisation of the study
The study was coordinated from ECVAM. A Management Team (MT) was constituted by four representatives of « lead laboratories », each of them being responsible for one of the four tests being evaluated.
The tests selected for inclusion in the validation study were the rat transcutaneous electrical resistance (TER) test, Corrositex TM, the Skin 2TM ZK1350 corrosivity test, and Episkin TM. Each test was conducted in three different laboratories, according to principles, criteria and procedures previously defined by ECVAM. Prediction models for each of the four tests were defined in the test protocols.
Coordination /MT /Laboratories
Sixty chemicals were selected by an independent Chemicals Selection Sub-Committee, and distributed coded to the participating laboratories. These included organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC), and soaps/surfactants (3NC). The selection is fully described in a publication (Ref. 1); the main criterion for including chemicals in the test set was that the corrosivity classifications were based on unequivocal animal data.
The results obtained were analysed by statistician experts. The classifications of the corrosivity potential of the test chemicals, as derived from the in vitro data obtained in the three laboratories conducting the test, were compared to the in vivo classifications independently assigned to the chemicals before the blind trial, to yield sensitivity, specificity, predictivity and accuracy of the test.
3- Main results
The full details of the validation study have been published (Ref. 2). Two tests, with a good reproductibility within and between test laboratories, proved applicable to the testing of a diverse group of chemicals : the TER test and Episkin.
In the TER test, test materials are applied for 2 to 24 hours to the epidermal surface of skin discs taken from the pelts of humanely killed young rats, and corrosive chemicals are identified by their ability to produce a loss of normal stratum corneum integrity, which is measured as a reduction of the inherent transcutaneous electrical resistance (below a predetermined threshold level).
Episkin is a tri-dimensional human skin model with a reconstructed epidermis and a functional stratum corneum. When utilised in corrosivity testing, application of test chemicals to the surface of the skin for 3, 60 and 240 min, is followed by an assessment of cell viability.
Sensitivity, specificity, predictivity and accuracy in distinguishing corrosive from non corrosive chemicals were very high for both tests: 88, 72, 72, 79 and 83, 80, 77, 81 % respectively for the TER test and Episkin. In addition, Episkin was also able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals
4- Opinion of the SCCNFP
ECVAM Scientific Advisory Committee (ESAC), which had been fully informed of the progression of the validation procedure, reviewed the final results and unanimously endorsed a statement that the rat skin TER test is scientifically validated for use as a replacement for the animal test for distinguishing between corrosive and non corrosive chemicals, and that Episkin is scientifically validated as a replacement for the animal test, and that these tests are ready for regulatory acceptance.
Sixty chemicals were used for the validation of these two methodologies; twenty of them are used as cosmetic ingredients, according to the "European inventory and common nomenclature of ingredients employed in cosmetic products" (Ref. 3).
SCCNFP reviewed publications from the validation study and ESAC statements, and propose that these two methods could be applied to the safety assessment of chemicals used as cosmetic ingredients.
A cosmetic ingredient or mixture of ingredients can be corrosive per se. When corrosivity cannot be excluded, testing for irritancy on animals or humans should be preceeded by a corrosivity test using one of these two validated in vitro methodologies.
5- References
1- Barratt M.D. & al. Toxicology in Vitro (1998) 12, 471-482
2- Barratt M.D. & al. Toxicology in vitro (1998) 12, 483-524
3- Commission Decision 96/335 EC of 8 May 1996 establishing an inventory and a common nomenclature of ingredients employed in cosmetic products J.O. L 132 of 1 June 1996
ANNEXE 2
SCCNFP/0069/98 Final - November 1998
OPINION ON IN VITRO METHODS TO ASSESS PHOTOTOXICITY IN THE SAFETY EVALUATION OF COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
Terms of reference
DG III requests the opinion of the Scientific Committee on Cosmetic and Non-Food Products (SCCNFP) as to the status of alternative methods for the safety assessment of cosmetic ingredients according to the current state of the art. Specifically DG III requests that the SCCNFP assesses the possibility of replacing data obtained on the basis of animal tests by data obtained making use of alternative methods in the safety evaluation of cosmetic ingredients (XXIV/1890/98)
1- Background
UV-absorbing chemicals are employed as ingredients of various cosmetic products. Guidelines for the safety testing of cosmetics require a test for photo-irritation potential of this type of compounds. Testing usually is done on animals, although an accepted protocol to test in vivo for photo-irritation potential does not exist.
2- Different phases in the study
2-1. In a first phase in 1992-1993, a joint EU/COLIPA prevalidation study was designed to identify in vitro test procedures for a validation trial under blind conditions. Twenty chemicals with known phototoxicity properties were selected according to scientific criteria by an independent COLIPA task force of experts. The chemicals underwent different tests e.g. photohaemolysis test, histidine oxidation test, Candida albicans test, SOLATEX PI ®, Skin 2®, Testskin ® and the 3T3 mouse fibroblast test. It came out that the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) with mouse fibroblasts using a sun simulator UV source (UVA 5J / cm 2) was giving better overall correlation to in vivo data than results from any of the other tests.
2-2. At ZEBET a prediction model for the 3T3 NRU PT test was developed, which took the IC-50 values from cytotoxicity dose-concentration curves in the presence and absence of exposure to UV-light into account. A photo-irritation factor (PIF) was calculated which is the ratio of IC 50 (-UV) / IC 50 (+UV).
Discriminant analysis showed that a PIF of 5.0 provided the best prediction to discriminate between phototoxic and non-phototoxic chemicals.
(Spielmann et al.1994b, 1995).
2-3. In the second phase in 1994-1995, the formal validation trial, the most promising in vitrohe validation study (Holzhütter 1997).
2-4. The results of the 3T3 NRU PT test were reproducible and correlated well with the in vivo data. Therefore, in 1997, the ECVAM Scientific Advisory Committee (ESAC) and in 1998 DG III and DG XI of the European Commission concluded from the formal validation study under blind conditions "that the 3T3 NRU PT is a well validated test and ready to be considered for regulatory acceptance" (Anon. 1998).
2-5. In 1996, the former Scientific Committee on Cosmetology (SCC) asked ECVAM to test the UV chemicals from Annex VII of the Directive 76/768/EEC in a blind trial using the 3T3 NRU PT test (XXIV/1878/97). The selection of the filters out of this list was done according to scientific criteria based on reliable in vivo data. (Guillot et al. 1985; Kaidbey and Kligman 1980). 8 UV filters were tested which were shown in vivo to be non phototoxic. To balance the study, 10 phototoxic and 10 non-phototoxic chemicals were tested under blind conditions in 4 laboratories; a correlation between 95 and 100 % was obtained when PIF or MPE, respectively, were used to predict the phototoxic potential and when concentrations between 0.1 and 10.0 µg/ml were tested. The management and the participants of this study concluded in 1998 (Spielmann et al. 1998 b) that the phototoxic potential of UV filters can be correctly assessed by the 3T3 NRU PT test.
2-6. In 1998, the SCCNFP reviewed carefully the publications from the validation studies, the ESAC statement and the application study of the UV filters. Critical questions were posed to the management team. These were all answered using appropriate scientific criteria.
3- Opinion of the SCCNFP
Taking the results obtained in the prevalidation and formal validation study of the 3T3 NRU PT test and the results of the application study of this test to the UV filters of Annex VII of the Directive 76/768/EEC into account, the SCCNFP proposes the use of the 3T3 NRU PT test as the standard method for testing the UV light absorbing cosmetic ingredients or mixtures of ingredients for phototoxic potential.
4- References
1. Spielmann H., Lovell W.W., Hölzle E., Johnson B.E., Maurer T., Miranda M.A., Pape W.J.W., Sapora O.H. & Sladowski D.
In vitro phototoxicity testing. The report and recommendations of ECVAM Workshop 2.
ATLA (1994 a) 22, 314-348.
2. Spielmann H., Balls M., Brand M., Döring B., Holzhütter H.G., Kalweit S., Klecak G., L'Eplattenier H., Liebsch M., Lovell W.W., Maurer T., Moldenhauer F., Moore L., Pape W.J.W., Pfannenbecker U., Potthast J., de Silva O., Steiling W. & Willshaw A.
EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a Balb/c 3T3 cell phototoxicity assay.
Toxicology in vitro (1994 b) 8, 793-796.
3. Spielmann H., Liebsh M., Pape W.J.W., Balls M., Dupuis J., Klecak G., Lovell W.W., Maurer T., de Silva O. & Steiling W.
EEC/COLIPA in vitro Photoirritancy Program : Results of the first stage of validation.
In : Elsner P., Maibach HI (eds) : Irritant Dermatitis. New Clinical and Experimental Aspects.
Curr. Probl. Dermatol. Basel, Karger (1995), vol. 23, pp 256-264.
4. EC - DG XXIV
Notes of guidance for testing of cosmetic ingredients for their safety evaluation. Annex 8.
(1997) XXIV/1878/97
5. Holzhütter H.G.
A general measure of in vitro phototoxicity derived from pairs of dose response curved and its use for predicting in vivo phototoxicity of chemicals.
ATLA (1997) 25, 445-462.
6. Spielmann H., Balls M., Dupuis J., Pape W.J.W., Pechovitch G.¸ de Silva O., Holzhütter H.G., Clothier R., Desolle P., Gerberick G.F., Liebsch M., Lovell W.W., Maurer T., Pfannenbecker U., Potthast J.M., Csato M., Sladowski D., Steiling W. & Brantom P.
The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU Phototoxicity test.
Toxicology in vitro (1998a) 12, 305-327.
7. Spielmann H., Balls M., Dupuis J., Pape W.J.W., de Silva O; Holzhütter H.G., Gerberick F., Liebsch M., Lovell W.W. & Pfannenbecker U.
A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test.
ATLA (1998 b) 26, 679-708.
8. Anon.
Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxic potential).
ATLA (1998) 26, 7-8.
9. Guillot J.P., Gonnet J.F., Loquerie J.F., Martin M.C., Convert P. & Cotte J. (1985).
A new method for the assessment of phototoxic and photoallergic potentials by topical applications in the albino guinea pig.
J Toxicol Cut & ocular toxicol. (1985) 4, 117-134.
10. Kaidbey K.H., Kligman A.M.
Photomaximisation tests for identifying photoallergic contact sensitisers.
Contact Dermatitis (1980) 6, 161-169.
11. Mandate for SCCNFP Specific Working Group on alternative methods, for the safety evaluation of cosmetic products (XXIV/1890/98) adopted on 20 May 1998.
ANNEXE 3
SCCNFP/0088/98 Final - January 1999
OPINION ON IN VITRO METHODS TO ASSESS PERCUTANEOUS ABSORPTION OF COSMETIC INGREDIENTS
Adopted by the SCCNFP at the plenary meeting of 20 January 1999
1. Background
In 1995, COLIPA ( European Cosmetic, Toiletry and Perfumery Association) presented to the former SCC (Scientific Committee on Cosmetics) Sub-Committee "Alternatives" an industrial view on the in vitro assessment of percutaneous absorption / penetration of cosmetic ingredients.
Guidelines for the testing of in vitro percutaneous absorption and some different protocols related to the use of excised skin (human, pig and rat) were proposed. Also, a general view on percutaneous absorption / penetration in vitro / in vivo correlation was presented based on a set of papers published in the scientific literature. The in vitro tests conducted by the cosmetic companies were developed to evaluate the safety of their cosmetic ingredients. They had not been intended for regulatory purposes and they were not subjected to the official validation processes.
The main conclusion of that Sub-Committee was the following (DGXXIV/1874/95): "The most important problem deduced from the documentation submitted is the absence of results and correlation data (in the protocols not in the references attached). The documentation should be implemented with intra- and inter-laboratory results obtained on percutaneous absorption of several strategic compounds (wide spectrum) as well as on correlation of in vitro / in vivo data. A more uniform presentation of in vitro percutaneous absorption methodology should be considered, taking into account the different protocols presented".
In 1996, a report and recommendations of ECVAM Workshop 13 about Methods for assessing percutaneous absorption was published (ATLA 24, 81-106, 1996).
In the last few years, two OECD Proposals to evaluate percutaneous absorption by invivo / in vitro methods have been presented. COLIPA members have upgraded their initial data submission as requested by the Extended Steering Committee of the OECD but to our knowledge the document has not been finalised by the organisation.
2. Position of the Scientific Committee on Cosmetics (SCC)/Scientific Committee on Cosmetic and Non-Food products (SCCNFP)
In the Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation (XXIV/1878/97) the former SCC emphasised that the test protocols used by industry were not subjected to a formal validation test and it recommended that the existing documentation must be supplemented, as regards intra- and inter- laboratory reproducibility, the influence of the vehicle on the release of the cosmetic ingredients and other technical and experimental details. However, the SCCNFP is convinced of the relevance of in vitro methods and has since recent years agreed to consider in vitro percutaneous absorption data in the evaluation of the safety of several cosmetic ingredients.
3. Submission of COLIPA data on in vitro/in vivo dermal absorption/percutaneous penetration (SCCNFP/0073/98)
In November 1998, COLIPA submitted a new document on in vitro / in vivo dermal absorption / percutaneous penetration including data and protocols used by several cosmetic companies.
These data refer to the dermal absorption / percutaneous absorption of chemical UV-filters, hair dyes (with rinsing or without rinsing) and several other ingredients.
In the methodologies used, the penetration cell design, the composition of the receptor fluid, the membrane integrity checking and the preparation of the dose of a given substance are described.
Experimental details concerning the application of test substance, reference chemicals, the fluid dynamics, temperature, exposure time, duration of the study, sampling and analytical techniques are also indicated.
Porcine back and flank skin, rat dorsal skin, guinea pig skin and human split-thickness skin have been used for the in vitro tests.
Some reference chemicals with a broad range of partition coefficient octanol/water (log P) and with different percutaneous absorption profiles have been evaluated. Benzoic acid, caffeine, estradiol. hydrocortisone, inulin, pentadecanoic acid, salicylic acid, sucrose, thiourea, tritiated water have been tested.
Among others these comparisons have been made:
Pig skin in vitro / Human skin in vivo (SC stripping)
Pig skin in vitro / Rat skin in vivo
Human skin in vitro / Pig skin in vitro
Some intra-assay reproducibility and inter-laboratory comparisons are included in the documentation. Additionally, in this document, information is included about the self- evaluation of each methodology according to the Canadian/US proposal for the Data Submission Form (OECD).
4. Opinion of the SCCNFP
The SCCNFP has reviewed the documentation submitted by COLIPA and agrees with the rationale for using in vitro methods to evaluate the dermal absorption / percutaneous penetration of cosmetic ingredients. The data reported in this document indicates the possible usefulness of the in vitro methodologies.
However the data provided at the moment are not sufficient to formulate a scientific opinion on how to conduct in vitro percutaneous absorption studies and assess the results.
The minimal requirements needed for the acceptance of in vitro percutaneous absorption studies to be evaluated, will be formulated by the SCCNFP, based on the scientific literature and on the experience of the Committee in evaluating the dossiers submitted for inclusion of cosmetic ingredients in the annexes of the Cosmetics Directive 76/768/EEC.
Studies to standardise methodologies for in vitro percutaneous absorption for cosmetic ingredients are necessary and the methods should be shown to give reproducible and relevant results. It is recommended that independent research institutes should perform or co-ordinate this work.
ANNEXE 4
SCCNFP/0003/98 Final - November 1998
OPINION - GUIDELINES ON THE USE OF HUMAN VOLUNTEERS IN THE TESTING OF POTENTIALLY CUTANEOUS IRRITANT COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
1 Background
1.1 Emphasis on consumer safety
According to the Council Directive "a cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use" (76/768/EEC). In order to achieve this goal of product safety, toxicological data on cosmetic ingredients are needed as outlined in the SCCNFP Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation 2 nd Rev. (XXIV/1878/97). Among the data mentioned, also «human data» are cited. However, the document does not specify these in detail. Regarding skin irritation, the SCCNFP considers that at present human testing of cosmetic ingredients or mixtures of ingredients should not be preferred to animal testing.
1.2 Animal tests for assessment of safety to be replaced by alternative methods
In the past, most of the toxicological data mentioned above have been generated by testing on animals. However, according to Council Directive 76/768/EEC, the marketing of cosmetic products containing ingredients or combinations of ingredients tested on animals after 30 June 2000 in order to meet the requirements of this Directive shall be prohibited. The Commission's general policy regarding research on animals supports the development of alternative methods to reduce or replace animal testing when possible.
1.3 Testing of cosmetic ingredients in humans
In this context, the scientific and ethical considerations for testing cosmetic ingredients or mixtures of ingredients in human subjects need to be defined more clearly. The skin irritancy reaction in humans is not an absolute measure and must be related to appropriate controls defining the range of response.
The SCCNFP stresses three points:
1. Since tests in animals or validated alternative methods may be limited regarding their predictive value for exposure of a human population, confirmatory safety tests in humans may be necessary scientifically and ethically, provided that the toxicological profile of an ingredient or a mixture of ingredients based on animal or alternative methods is available and that a high degree of safety is to be expected.
2. Confirmatory tests of ingredients or mixtures of ingredients in humans must be limited to situations where no irreversible damaging effects are to be expected for the volunteers and where the study goal is reasonably achievable with a study population of limited size.
3. The recruitment of human volunteers should be in line with the "World Medical Doctors Association Declaration of Helsinki" and "the Good Clinical Practice for trials on Medicinal Products in the European Community."
2 Procedure of irritancy assessment
The following text outlines the steps of an assessment of the irritancy of an ingredient or mixture of ingredients. While this text focuses on irritancy, it is understood that other aspects of toxicity have to be considered in parallel before performing tests in humans.
2.1 Initial considerations
Available chemical and physico-chemical data and structure-activity relationships making use of computer programs and databases for the prediction of skin irritation potential should be used.
2.2 Evaluation of irritation
Ingredients or mixtures of ingredients should be tested on animals and humans only at non-corrosive concentrations. This decision may be based on pH and acid/alkaline reserve measurements and on in vitro tests for skin corrosivity. At the present, in vitro methods for the assessment of irritancy have not yet been validated.
2.3 Confirmation by human volunteer testing
On the basis of a low irritation potential as proven by animal or future validated in vitro methods, the skin tolerability of an ingredient or a mixture of ingredient can be confirmed by testing in human volunteers. A number of test protocols are available such as open and closed patch tests, single and repeated-exposure tests. They should be chosen on the basis of the relevant use pattern of the ingredient or mixture of ingredients (1).
While these tests historically have been assessed by clinical methods, non-invasive bioengineering technology such as measurement of transepidermal water loss or of blood flow may provide higher sensitivity and objectivity of these tests and thereby reduce the exposure and risk to volunteers.
However, neither the above confirmatory tests nor the use of bioengineering methods have been validated according to modern scientific criteria. The SCCNFP recommends the Commission to support further research in this area.
2.4 Consumer market surveillance
The evaluation of irritation of an ingredient or mixture of ingredients is not finished with the introduction of respective cosmetic products on the market, but it should continue by making use of data generated by consumer market surveillance and other sources.
3 Ethical considerations
Confirmatory skin tolerance tests of cosmetic ingredients in humans are subject to ethical concerns. In order to take account of these concerns, to minimise the risk to volunteers and to safeguard their rights, test protocols should be submitted to an acknowledged ethical committee and be in compliance with the followings :
i) to supplement non-clinical information,
ii) to confirm that exposure will not cause significant harm, and/or,
iii) in a controlled fashion that minimises subject risk (4).
The investigator(s) in skin tolerability tests of cosmetic ingredients should fulfil the qualifications as mentioned in the CPMP Working Party on Efficacy of Medicinal Products Note for Guidance on Good Clinical Practice (3).
References
(1) Patrick E, Maibach HI: Predictive assays: Animal and man, and in vitro and in vivo. In: Rycroft R.J.G., Menne T., Frosch P.J., (1995) Textbook of Contact Dermatitis. Springer Heidelberg New York.
(2) World Medical Doctors Association Declaration of Helsinki, (1997) JAMA 227: 925-926
(3) CPMP Working Party on Efficacy of Medicinal Products Note for Guidance: Good Clinical Practice for Trials on Medicinal Products in the European Community (1990) CB-55-89-706-EN-C.
(4) Organisation for Economic Co-operation and Development: Development of OECD Test Guidelines for Use in Tests with Human Volunteers. 27 th Joint Meeting of the Chemicals Group and Management Committee, 11 th-13 th February 1998 (ENV/MC/CHEM/RD (98))
ANNEXE 5
SCCNFP/0004/98
Notes of Guidance for testing of Cosmetic Ingredients for their Safety Evaluation
Revision of Annex 7 : Microbiological Quality of the Finished Cosmetic Product
adopted by the plenary session of the SCCNFP of 23 September 1998
1. Preamble.
Skin and mucous membranes are normally protected from microbial attack by a natural mechanical barrier and defence mechanisms. However, protective integuments may be damaged and slight trauma may be caused by the action of some cosmetics that may enhance microbial infection. These situations may be of particular concern when cosmetics are used in the eye area or on mucous membranes or on damaged skin and when used by children under 3 years, elderly people and people showing compromised immune responses. These are the reasons to define two separate categories of cosmetic products in the microbiological quality control limits.
Although a very low number of cases of contamination in cosmetics leading to microbial infections have been reported, it is likely that under-reported clinical microbiological problems (for instance infectious folliculites) associated to the use of contaminated cosmetics are recognised by several dermatologists (to be reported in a separate document). On the other hand microbial contamination may spoil cosmetic products or reduce the intended quality. These statements make it necessary to carry out routine microbiological control of cosmetics, in order to ensure their quality and the safety for customers to use.
2. Categories of cosmetics in microbiological quality control.
In relation with the microbiological quality control, two categories of cosmetics are defined.
Category 1: Products specifically intended for children under 3 years, eye area and
mucous membranes.
Category 2: Other products.
3. Quantitative limits.
The limit for cosmetics classified in Category 1 is: total viable count for aerobic mesophyllic micro-organisms not more than 10 2 cfu/g or ml in 0.5 g or ml of the product.
The limit for cosmetics classified in Category 2 is: total viable count for aerobic mesophyllic micro-organisms not more than 10 3 cfu/g or ml in 0.1 g or ml of the product.
4. Qualitative limits.
Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans are considered the main potential pathogens in cosmetic products. These specified potential pathogens must not be detectable in 0.5 g or ml of the cosmetic product in cosmetics of Category 1 and in 0.1 g or ml in cosmetics of Category 2.
5. Product preservation.
Microbial contaminants have two origins: during production and filling, and during the use of the cosmetic by the customer. From the moment in which the cosmetic unit is opened until the consumer finishes the product, there is a permanent, variable and additive microbial contamination of the cosmetic caused by the domestic environment and the consumer's body (hands and body skin). The reasons for the need of microbial preservation in cosmetics are the following:
5.1. To ensure the microbial safety of cosmetics for customers to use.
5.2. To maintain the quality and specifications intended for the product.
5.3. To confirm hygienic and high-quality handling.
6. The challenge testing
The efficacy of the preservation has to be assessed experimentally during the development process to ensure microbial stability and preservation by challenge testing. Challenge testing is mandatory for all those products that in normal conditions of storage and use, a risk of infection for the consumer or a deterioration of the product exist. The challenge test consists of an artificial contamination of the finished product and a posterior evaluation of the decrease of this contamination to levels ensuring the microbial limits established in products of Category 1 and 2.
The micro-organisms used in the challenge test will be issued from official collection strains from any state in the EU to ensure reproducibility of the test and will be: Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Additional bacteria and fungi might be used for additional specific purposes of the challenge testing. The microcidal activity of preservatives or any other compound in the finished cosmetic must be ruled out in the challenge test by dilution, filtration, neutralisers or any other means. The experimental performance of the microbial controls and the challenge tests must be laid down and validated by a microbiologist.
7. Good Manufacturing Practice.
In order to accomplish with the Good Manufacturing Practices and Microbial Quality Management, manufacturers of cosmetics have to define and follow specific cleaning, sanitation and control procedures to keep appropriately clean and free of micro-organisms that could be harmful for the consumers or adverse for the quality of the cosmetics. These proceedings will include procedures to microbiology control raw materials, bulk and finished products, packaging components, personnel, equipment and locals.
The opinion concerns :
a- the status of alternative methods to animal testing for the safety assessment of cosmetic ingredients in January 1999. The SCCNFP is of the opinion that three in vitro methods to assess skin corrosivity and phototoxicity have been validated and can be considered applicable to the safety evaluation of cosmetic ingredients or mixtures of ingredients. These methods are the "Rat Skin Transcutaneous Electrical Resistance Test" and the "EPISKIN Test" for the assessment of skin corrosivity, and the "3T3 Neutral Red Uptake Phototoxicity Test" for the assessment of phototoxicity.
The SCCNFP has considered the possible usefulness of the various existing in vitro methodologies to evaluate the percutaneous absorption of cosmetic ingredients and will define a set of minimal criteria needed for the acceptance of in vitro percutaneous absorption studies for the evaluation of toxicological profile of cosmetic ingredients.
b- the updating of the Notes of Guidance (XXIV/1878/97) which includes guidelines on the use of human volunteers in the testing of potentially cutaneous irritant cosmetic ingredients or mixtures of ingredients and an opinion on the microbiological quality of finished cosmetic products.
c- the issues regarding alternative methods and the dossier to be discussed during the year 1999.
1.Mandate to the SCCNFP (XXIV/1890/98)
1.1 The SCCNFP shall act as a resource of scientific expertise to the European Commission, with regards to the development of alternative methods. To that effect they will regularly meet with representatives from the concerned parties to :
However, such advice in no way prejudices the right of the SCCNFP to reject data previously discussed, if the scientific standard of the work is considered inappropriate.
Although this mandate has been developed in relation to cosmetic ingredients, it is envisaged that scientifically sound non-animal methods for safety assessment will have broad application.
1.2. The SCCNFP was requested to continuously update the Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation as stated on page 8 of the 2 nd Revision, adopted by the former SCC (Scientific Committee on Cosmetology) on January 16 th, 1997.
Due to the almost complete review of the former SCC, and the presence in the SCCNFP of experts on other disciplines, it was decided to include technical modifications to the content of the Notes, such as Guidelines for Human Testing, as it seems appropriate to give advice on this new aspects.
2. Background
In a note from the responsible Commission Service of August 11 th 1998, with the subject to review on the status of alternative methods to animal testing in the safety evaluation of cosmetic ingredients, it is stated:
"... it would be appreciated if the summary on the status of alternative methods for safety assessment of cosmetic ingredients could be available by the end of January 1999 at the latest".
The Specific Working Party (SPW) of the SCCNFP "Alternatives to Animal Testing & Dossiers" was established on November 14 th 1997 (during the 1 st Plenary Meeting of the SCCNFP) and was put into operation in January 1998. The SPW met on Feb. 16 th, March 4-5 th, March 24-25 th, April 20-21 st, May 12-13 th, June 16 th, Sept. 22 nd, Nov. 24 th, Dec. 10 th. It met with representatives of the concerned Commission Services, of ECVAM (European Center for the Validation of Alternative Methods) and with representatives of the European Cosmetic Industry.
3.Opinion on the Use of Alternative Methods to Animal Testing in the Safety Evaluation of Cosmetic Ingredients or Mixture of Ingredients
3.1. In Vitro Methods to Assess Skin Corrosivity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients
ECVAM has concluded positively the validation of two in vitro methods to assess the skin corrosivity potential of several chemicals, including 60 agents employed as cosmetic ingredients.
The SCCNFP considers that in the safety evaluation of chemicals intended to be used as cosmetic ingredients, when corrosivity potential cannot be excluded, they should be tested by the "Rat Skin Transcutaneous Electrical Resistance (TER) Test" or by the "EPISKIN Test", before testing for irritancy on animals or humans. An opinion of the SCCNFP has been adopted during its Plenary Session on November 25 th, 1998 (SCCNFP/0070/98Final, November 1998).
The guideline on the use of TER and EPISKIN methods has been presented by ECVAM to OECD (98/III/COS/21) and it is now ready for approval. Annex 1.
3.2. In Vitro Methods to Assess Phototoxicity in the Safety Evaluation of Cosmetic Ingredients or Mixtures of Ingredients
As reported in the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97) adopted by the former SCC, all chemicals which can absorb UVA and/or UVB light may change their molecular configuration and may undergo further biological reactions of toxicological relevance for consumers. The former SCC as well as the present SCCNFP request for all such chemicals routine testing for phototoxicity.
Animal models have not been validated for testing for phototoxicity.
ECVAM, in cooperation with COLIPA (European Cosmetic, Toilettry and Perfumery Association), has concluded a series of studies representing pre-validation, validation and application to cosmetic ingredients of one in vitro method, named "3T3 Neutral Red Uptake, Phototoxicity Test (3T3 NRU PT)".
The principle of the method is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of UVA/visible light.
The predictive value of this method for a potential human phototoxic chemical has been demonstrated to be between 95 and 100%.
The method has been demonstrated to be able to identify UV filter chemicals which are non-phototoxic for consumers and which are employed in cosmetic products. A guideline for testing UV light absorbing chemicals by using 3T3 NRU PT has been presented to OECD.
An opinion of the SCCNFP has been adopted during its Plenary Session on November 25 th, 1998, proposing the use of 3T3 NRU PT test as a method for testing UV light absorbing cosmetic ingredients, or mixtures of ingredients for phototoxic potential (SCCNFP/0069/98 Final, November 1998): Annex 2.
3.3. In Vitro Methods to Assess Percutaneous Absorption of Cosmetic Ingredients
The assessment of the percutaneous absorption of a given cosmetic ingredient has primary relevance in the procedure for evaluating the safety of cosmetics for consumers. The need to develop a series of toxicological studies as a new cosmetic ingredient depends very much on the knowledge of the percutaneously absorbed dose which could be distributed systemically in the human body (XXIV/1878/97).
In the last few years, two OECD Proposals to evaluate percutaneous absorption by in vivo or in vitro methods have been presented, but none of them has been accepted in the regulatory system yet.
COLIPA has presented a technical document on in vitro/ in vivo percutaneous absorption, reporting data and protocols in use since many years by several cosmetic companies, in the evaluation of the safety of hair dyes, UV filters and some other cosmetic ingredients.
The data presented include technical information on methodologies used and contain technical details in relation to the systems developed to assess percutaneous absorption, the application of the test substance and the conditions during the analysis, the type of the skin and the species from which it derives, a series of comparisons between different species of skin and different systems (namely in vitro Vs in vivo) some intra-assay and inter-laboratory comparisons, the evaluation of the results.
The SCCNFP has reviewed this documentation submitted by COLIPA and agrees with the rationale for using in vitro methods to evaluate the percutaneous absorption as it was already recommended in the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97).
However, the SCCNFP considers relevant to define in a short time a set of minimal criteria needed for the acceptance of in vitro percutaneous absorption studies to be evaluated when the dossiers of these cosmetic ingredients for their inclusion in the Annexes of Cosmetic Directive 76/768/EEC are submitted.
An opinion of the SCCNFP was adopted during its Plenary Meeting on January 20 th 1999, proposing the use of in vitro methods to assess percutaneous absorption of cosmetic ingredients (SCCNFP/0088/98 Rev 2, January 1999): Annex 3.
4. Updating of Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation
4.1. Guidelines on the Use of Human Volunteers in the Testing of Potentially Cutaneous Irritant Cosmetic Ingredients or Mixtures of Ingredients
In the "Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation" (XXIV/1878/97) the former SCC stated that "for an analysis of potential adverse effects of a cosmetic product or ingredient (e.g. irritation, sensitization, non-invasive cutaneous penetration studies) observations on human subjects should be used if available". This opinion was recently expressed also by the present SCCNFP, by considering that tests on animals for skin irritation or (not yet) validated alternative methods may be limited regarding their predictive value for exposure of a human population. The SCCNFP states that confirmatory tests on humans should be scientifically and ethically necessary, provided that the toxicological profile of an ingredient or a mixture of ingredients based on animal or alternative methods is available and that a high degree of safety is to be expected (SCCNFP/0003/98). Therefore, an opinion on the use of human volunteers in the testing of potentially cutaneous irritant cosmetic ingredients has been adopted by the SCCNFP in its plenary session of November 25 th, 1998 (SCCNFP/0003/98 Final, November 1998): Annex 4.
This document presents the general consensus of the SCCNFP regarding one aspect of human testing, however stating again that, at present, human testing of cosmetic ingredients should not be preferred to animal testing and that the safety testing of cosmetics on humans may not be considered to be an alternative method to the use of animals.
The present opinion also stresses the concept that confirmatory skin tolerance tests of cosmetics in humans are subject to ethical concern. According to the Declaration of Helsinki "safety studies of cosmetics in human must be subjected to National Regulations and Good Clinical Practice for Trials on Medicinal Products in the European Union".
Test protocols to be applied in the safety evaluation of cosmetic ingredients or their mixtures with human studies have not been validated yet. SCCNFP's opinion is that a set of specific guidelines concerning the most applied protocols can be prepared in the near future and presented to the European Commission. The SCNFP has been requested by the responsible Commission Service to offer guidance on:
A draft proposal of "Guidelines on the Testing of Cosmetic Finished Products on Human Volunteers" is currently under preparation and is being discussed by the SCCNFP. An opinion should soon be available.
4.2. Revision of Annex 7 of Notes of Guidance. Microbiological Quality of Finished Cosmetic Products (XXIV/1878/97)
As stated several times by the former SCC, the "Notes of Guidance..." will require future amendments as scientific knowledge advances. A second reason for revising the document will depend on the improvement of cosmetic technologies in the industrial sector.
On September 23 rd 1998 the SCCNFP has adopted the Revision of Annex 7. "Microbiological Quality of the Finished Products", as amendment to the previous document (SCCNFP/0004/98 Final). Annex 5.
5. Work Programme for 1999
The work planned for 1999 in fulfillment of the Commission's request is schematically represented by the following list:
ANNEX 1
SCCNFP/0070/98 Final - November 1998
The Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers
OPINION ON IN VITRO METHODS TO ASSESS SKIN CORROSIVITY IN THE SAFETY EVALUATION OF COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
Terms of Reference
Two in vitro methods developed to assess skin corrosivity of chemicals, the "Rat skin Trancutaneous Electrical Resistance (TER)test" and the "EPISKIN test" have been validated by ESAC (ECVAM Scientific Advisory Committee).
The Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) has been requested by DG III to advise the Commission on the applicability of the methods to the safety assessment of chemicals used as cosmetic ingredients.
1- Background
The European Centre for the Validation of Alternative Methods (ECVAM) has conducted in 1996-1997 a validation study of in vitro tests developed to assess skin corrosivity of chemicals. This study was a follow-up to a pre-validation study of tests developed for replacing the in vivo Draize skin corrosivity test in rabbits.
The main objectives of the validation study, as defined by the sponsors and the management team before the study began, were :
(a) to identify tests capable of discriminating corrosives (C) from non corrosive (NC) for selected groups of chemicals (e.g. organic acids, phenols) and/or all chemicals (single chemical entities only);
(b) to determine whether the tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals.
2- Organisation of the study
The study was coordinated from ECVAM. A Management Team (MT) was constituted by four representatives of « lead laboratories », each of them being responsible for one of the four tests being evaluated.
The tests selected for inclusion in the validation study were the rat transcutaneous electrical resistance (TER) test, Corrositex TM, the Skin 2TM ZK1350 corrosivity test, and Episkin TM. Each test was conducted in three different laboratories, according to principles, criteria and procedures previously defined by ECVAM. Prediction models for each of the four tests were defined in the test protocols.
Coordination /MT /Laboratories
Sixty chemicals were selected by an independent Chemicals Selection Sub-Committee, and distributed coded to the participating laboratories. These included organic acids (6C/5NC), organic bases (7C/3NC), neutral organics (9NC), phenols (2C/3NC), inorganic acids (6C/1NC), inorganic bases (2C/2NC), inorganic salts (1C/2NC), electrophiles (3C/5NC), and soaps/surfactants (3NC). The selection is fully described in a publication (Ref. 1); the main criterion for including chemicals in the test set was that the corrosivity classifications were based on unequivocal animal data.
The results obtained were analysed by statistician experts. The classifications of the corrosivity potential of the test chemicals, as derived from the in vitro data obtained in the three laboratories conducting the test, were compared to the in vivo classifications independently assigned to the chemicals before the blind trial, to yield sensitivity, specificity, predictivity and accuracy of the test.
3- Main results
The full details of the validation study have been published (Ref. 2). Two tests, with a good reproductibility within and between test laboratories, proved applicable to the testing of a diverse group of chemicals : the TER test and Episkin.
In the TER test, test materials are applied for 2 to 24 hours to the epidermal surface of skin discs taken from the pelts of humanely killed young rats, and corrosive chemicals are identified by their ability to produce a loss of normal stratum corneum integrity, which is measured as a reduction of the inherent transcutaneous electrical resistance (below a predetermined threshold level).
Episkin is a tri-dimensional human skin model with a reconstructed epidermis and a functional stratum corneum. When utilised in corrosivity testing, application of test chemicals to the surface of the skin for 3, 60 and 240 min, is followed by an assessment of cell viability.
Sensitivity, specificity, predictivity and accuracy in distinguishing corrosive from non corrosive chemicals were very high for both tests: 88, 72, 72, 79 and 83, 80, 77, 81 % respectively for the TER test and Episkin. In addition, Episkin was also able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals
4- Opinion of the SCCNFP
ECVAM Scientific Advisory Committee (ESAC), which had been fully informed of the progression of the validation procedure, reviewed the final results and unanimously endorsed a statement that the rat skin TER test is scientifically validated for use as a replacement for the animal test for distinguishing between corrosive and non corrosive chemicals, and that Episkin is scientifically validated as a replacement for the animal test, and that these tests are ready for regulatory acceptance.
Sixty chemicals were used for the validation of these two methodologies; twenty of them are used as cosmetic ingredients, according to the "European inventory and common nomenclature of ingredients employed in cosmetic products" (Ref. 3).
SCCNFP reviewed publications from the validation study and ESAC statements, and propose that these two methods could be applied to the safety assessment of chemicals used as cosmetic ingredients.
A cosmetic ingredient or mixture of ingredients can be corrosive per se. When corrosivity cannot be excluded, testing for irritancy on animals or humans should be preceeded by a corrosivity test using one of these two validated in vitro methodologies.
5- References
1- Barratt M.D. & al. Toxicology in Vitro (1998) 12, 471-482
2- Barratt M.D. & al. Toxicology in vitro (1998) 12, 483-524
3- Commission Decision 96/335 EC of 8 May 1996 establishing an inventory and a common nomenclature of ingredients employed in cosmetic products J.O. L 132 of 1 June 1996
ANNEXE 2
SCCNFP/0069/98 Final - November 1998
OPINION ON IN VITRO METHODS TO ASSESS PHOTOTOXICITY IN THE SAFETY EVALUATION OF COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
Terms of reference
DG III requests the opinion of the Scientific Committee on Cosmetic and Non-Food Products (SCCNFP) as to the status of alternative methods for the safety assessment of cosmetic ingredients according to the current state of the art. Specifically DG III requests that the SCCNFP assesses the possibility of replacing data obtained on the basis of animal tests by data obtained making use of alternative methods in the safety evaluation of cosmetic ingredients (XXIV/1890/98)
1- Background
UV-absorbing chemicals are employed as ingredients of various cosmetic products. Guidelines for the safety testing of cosmetics require a test for photo-irritation potential of this type of compounds. Testing usually is done on animals, although an accepted protocol to test in vivo for photo-irritation potential does not exist.
2- Different phases in the study
2-1. In a first phase in 1992-1993, a joint EU/COLIPA prevalidation study was designed to identify in vitro test procedures for a validation trial under blind conditions. Twenty chemicals with known phototoxicity properties were selected according to scientific criteria by an independent COLIPA task force of experts. The chemicals underwent different tests e.g. photohaemolysis test, histidine oxidation test, Candida albicans test, SOLATEX PI ®, Skin 2®, Testskin ® and the 3T3 mouse fibroblast test. It came out that the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) with mouse fibroblasts using a sun simulator UV source (UVA 5J / cm 2) was giving better overall correlation to in vivo data than results from any of the other tests.
2-2. At ZEBET a prediction model for the 3T3 NRU PT test was developed, which took the IC-50 values from cytotoxicity dose-concentration curves in the presence and absence of exposure to UV-light into account. A photo-irritation factor (PIF) was calculated which is the ratio of IC 50 (-UV) / IC 50 (+UV).
Discriminant analysis showed that a PIF of 5.0 provided the best prediction to discriminate between phototoxic and non-phototoxic chemicals.
(Spielmann et al.1994b, 1995).
2-3. In the second phase in 1994-1995, the formal validation trial, the most promising in vitrohe validation study (Holzhütter 1997).
2-4. The results of the 3T3 NRU PT test were reproducible and correlated well with the in vivo data. Therefore, in 1997, the ECVAM Scientific Advisory Committee (ESAC) and in 1998 DG III and DG XI of the European Commission concluded from the formal validation study under blind conditions "that the 3T3 NRU PT is a well validated test and ready to be considered for regulatory acceptance" (Anon. 1998).
2-5. In 1996, the former Scientific Committee on Cosmetology (SCC) asked ECVAM to test the UV chemicals from Annex VII of the Directive 76/768/EEC in a blind trial using the 3T3 NRU PT test (XXIV/1878/97). The selection of the filters out of this list was done according to scientific criteria based on reliable in vivo data. (Guillot et al. 1985; Kaidbey and Kligman 1980). 8 UV filters were tested which were shown in vivo to be non phototoxic. To balance the study, 10 phototoxic and 10 non-phototoxic chemicals were tested under blind conditions in 4 laboratories; a correlation between 95 and 100 % was obtained when PIF or MPE, respectively, were used to predict the phototoxic potential and when concentrations between 0.1 and 10.0 µg/ml were tested. The management and the participants of this study concluded in 1998 (Spielmann et al. 1998 b) that the phototoxic potential of UV filters can be correctly assessed by the 3T3 NRU PT test.
2-6. In 1998, the SCCNFP reviewed carefully the publications from the validation studies, the ESAC statement and the application study of the UV filters. Critical questions were posed to the management team. These were all answered using appropriate scientific criteria.
3- Opinion of the SCCNFP
Taking the results obtained in the prevalidation and formal validation study of the 3T3 NRU PT test and the results of the application study of this test to the UV filters of Annex VII of the Directive 76/768/EEC into account, the SCCNFP proposes the use of the 3T3 NRU PT test as the standard method for testing the UV light absorbing cosmetic ingredients or mixtures of ingredients for phototoxic potential.
4- References
1. Spielmann H., Lovell W.W., Hölzle E., Johnson B.E., Maurer T., Miranda M.A., Pape W.J.W., Sapora O.H. & Sladowski D.
In vitro phototoxicity testing. The report and recommendations of ECVAM Workshop 2.
ATLA (1994 a) 22, 314-348.
2. Spielmann H., Balls M., Brand M., Döring B., Holzhütter H.G., Kalweit S., Klecak G., L'Eplattenier H., Liebsch M., Lovell W.W., Maurer T., Moldenhauer F., Moore L., Pape W.J.W., Pfannenbecker U., Potthast J., de Silva O., Steiling W. & Willshaw A.
EEC/COLIPA project on in vitro phototoxicity testing: first results obtained with a Balb/c 3T3 cell phototoxicity assay.
Toxicology in vitro (1994 b) 8, 793-796.
3. Spielmann H., Liebsh M., Pape W.J.W., Balls M., Dupuis J., Klecak G., Lovell W.W., Maurer T., de Silva O. & Steiling W.
EEC/COLIPA in vitro Photoirritancy Program : Results of the first stage of validation.
In : Elsner P., Maibach HI (eds) : Irritant Dermatitis. New Clinical and Experimental Aspects.
Curr. Probl. Dermatol. Basel, Karger (1995), vol. 23, pp 256-264.
4. EC - DG XXIV
Notes of guidance for testing of cosmetic ingredients for their safety evaluation. Annex 8.
(1997) XXIV/1878/97
5. Holzhütter H.G.
A general measure of in vitro phototoxicity derived from pairs of dose response curved and its use for predicting in vivo phototoxicity of chemicals.
ATLA (1997) 25, 445-462.
6. Spielmann H., Balls M., Dupuis J., Pape W.J.W., Pechovitch G.¸ de Silva O., Holzhütter H.G., Clothier R., Desolle P., Gerberick G.F., Liebsch M., Lovell W.W., Maurer T., Pfannenbecker U., Potthast J.M., Csato M., Sladowski D., Steiling W. & Brantom P.
The international EU/COLIPA in vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU Phototoxicity test.
Toxicology in vitro (1998a) 12, 305-327.
7. Spielmann H., Balls M., Dupuis J., Pape W.J.W., de Silva O; Holzhütter H.G., Gerberick F., Liebsch M., Lovell W.W. & Pfannenbecker U.
A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test.
ATLA (1998 b) 26, 679-708.
8. Anon.
Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxic potential).
ATLA (1998) 26, 7-8.
9. Guillot J.P., Gonnet J.F., Loquerie J.F., Martin M.C., Convert P. & Cotte J. (1985).
A new method for the assessment of phototoxic and photoallergic potentials by topical applications in the albino guinea pig.
J Toxicol Cut & ocular toxicol. (1985) 4, 117-134.
10. Kaidbey K.H., Kligman A.M.
Photomaximisation tests for identifying photoallergic contact sensitisers.
Contact Dermatitis (1980) 6, 161-169.
11. Mandate for SCCNFP Specific Working Group on alternative methods, for the safety evaluation of cosmetic products (XXIV/1890/98) adopted on 20 May 1998.
ANNEXE 3
SCCNFP/0088/98 Final - January 1999
OPINION ON IN VITRO METHODS TO ASSESS PERCUTANEOUS ABSORPTION OF COSMETIC INGREDIENTS
Adopted by the SCCNFP at the plenary meeting of 20 January 1999
1. Background
In 1995, COLIPA ( European Cosmetic, Toiletry and Perfumery Association) presented to the former SCC (Scientific Committee on Cosmetics) Sub-Committee "Alternatives" an industrial view on the in vitro assessment of percutaneous absorption / penetration of cosmetic ingredients.
Guidelines for the testing of in vitro percutaneous absorption and some different protocols related to the use of excised skin (human, pig and rat) were proposed. Also, a general view on percutaneous absorption / penetration in vitro / in vivo correlation was presented based on a set of papers published in the scientific literature. The in vitro tests conducted by the cosmetic companies were developed to evaluate the safety of their cosmetic ingredients. They had not been intended for regulatory purposes and they were not subjected to the official validation processes.
The main conclusion of that Sub-Committee was the following (DGXXIV/1874/95): "The most important problem deduced from the documentation submitted is the absence of results and correlation data (in the protocols not in the references attached). The documentation should be implemented with intra- and inter-laboratory results obtained on percutaneous absorption of several strategic compounds (wide spectrum) as well as on correlation of in vitro / in vivo data. A more uniform presentation of in vitro percutaneous absorption methodology should be considered, taking into account the different protocols presented".
In 1996, a report and recommendations of ECVAM Workshop 13 about Methods for assessing percutaneous absorption was published (ATLA 24, 81-106, 1996).
In the last few years, two OECD Proposals to evaluate percutaneous absorption by invivo / in vitro methods have been presented. COLIPA members have upgraded their initial data submission as requested by the Extended Steering Committee of the OECD but to our knowledge the document has not been finalised by the organisation.
2. Position of the Scientific Committee on Cosmetics (SCC)/Scientific Committee on Cosmetic and Non-Food products (SCCNFP)
In the Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation (XXIV/1878/97) the former SCC emphasised that the test protocols used by industry were not subjected to a formal validation test and it recommended that the existing documentation must be supplemented, as regards intra- and inter- laboratory reproducibility, the influence of the vehicle on the release of the cosmetic ingredients and other technical and experimental details. However, the SCCNFP is convinced of the relevance of in vitro methods and has since recent years agreed to consider in vitro percutaneous absorption data in the evaluation of the safety of several cosmetic ingredients.
3. Submission of COLIPA data on in vitro/in vivo dermal absorption/percutaneous penetration (SCCNFP/0073/98)
In November 1998, COLIPA submitted a new document on in vitro / in vivo dermal absorption / percutaneous penetration including data and protocols used by several cosmetic companies.
These data refer to the dermal absorption / percutaneous absorption of chemical UV-filters, hair dyes (with rinsing or without rinsing) and several other ingredients.
In the methodologies used, the penetration cell design, the composition of the receptor fluid, the membrane integrity checking and the preparation of the dose of a given substance are described.
Experimental details concerning the application of test substance, reference chemicals, the fluid dynamics, temperature, exposure time, duration of the study, sampling and analytical techniques are also indicated.
Porcine back and flank skin, rat dorsal skin, guinea pig skin and human split-thickness skin have been used for the in vitro tests.
Some reference chemicals with a broad range of partition coefficient octanol/water (log P) and with different percutaneous absorption profiles have been evaluated. Benzoic acid, caffeine, estradiol. hydrocortisone, inulin, pentadecanoic acid, salicylic acid, sucrose, thiourea, tritiated water have been tested.
Among others these comparisons have been made:
Pig skin in vitro / Human skin in vivo (SC stripping)
Pig skin in vitro / Rat skin in vivo
Human skin in vitro / Pig skin in vitro
Some intra-assay reproducibility and inter-laboratory comparisons are included in the documentation. Additionally, in this document, information is included about the self- evaluation of each methodology according to the Canadian/US proposal for the Data Submission Form (OECD).
4. Opinion of the SCCNFP
The SCCNFP has reviewed the documentation submitted by COLIPA and agrees with the rationale for using in vitro methods to evaluate the dermal absorption / percutaneous penetration of cosmetic ingredients. The data reported in this document indicates the possible usefulness of the in vitro methodologies.
However the data provided at the moment are not sufficient to formulate a scientific opinion on how to conduct in vitro percutaneous absorption studies and assess the results.
The minimal requirements needed for the acceptance of in vitro percutaneous absorption studies to be evaluated, will be formulated by the SCCNFP, based on the scientific literature and on the experience of the Committee in evaluating the dossiers submitted for inclusion of cosmetic ingredients in the annexes of the Cosmetics Directive 76/768/EEC.
Studies to standardise methodologies for in vitro percutaneous absorption for cosmetic ingredients are necessary and the methods should be shown to give reproducible and relevant results. It is recommended that independent research institutes should perform or co-ordinate this work.
ANNEXE 4
SCCNFP/0003/98 Final - November 1998
OPINION - GUIDELINES ON THE USE OF HUMAN VOLUNTEERS IN THE TESTING OF POTENTIALLY CUTANEOUS IRRITANT COSMETIC INGREDIENTS OR MIXTURES OF INGREDIENTS
Adopted by the plenary session of the SCCNFP of 25 November 1998
1 Background
1.1 Emphasis on consumer safety
According to the Council Directive "a cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use" (76/768/EEC). In order to achieve this goal of product safety, toxicological data on cosmetic ingredients are needed as outlined in the SCCNFP Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation 2 nd Rev. (XXIV/1878/97). Among the data mentioned, also «human data» are cited. However, the document does not specify these in detail. Regarding skin irritation, the SCCNFP considers that at present human testing of cosmetic ingredients or mixtures of ingredients should not be preferred to animal testing.
1.2 Animal tests for assessment of safety to be replaced by alternative methods
In the past, most of the toxicological data mentioned above have been generated by testing on animals. However, according to Council Directive 76/768/EEC, the marketing of cosmetic products containing ingredients or combinations of ingredients tested on animals after 30 June 2000 in order to meet the requirements of this Directive shall be prohibited. The Commission's general policy regarding research on animals supports the development of alternative methods to reduce or replace animal testing when possible.
1.3 Testing of cosmetic ingredients in humans
In this context, the scientific and ethical considerations for testing cosmetic ingredients or mixtures of ingredients in human subjects need to be defined more clearly. The skin irritancy reaction in humans is not an absolute measure and must be related to appropriate controls defining the range of response.
The SCCNFP stresses three points:
1. Since tests in animals or validated alternative methods may be limited regarding their predictive value for exposure of a human population, confirmatory safety tests in humans may be necessary scientifically and ethically, provided that the toxicological profile of an ingredient or a mixture of ingredients based on animal or alternative methods is available and that a high degree of safety is to be expected.
2. Confirmatory tests of ingredients or mixtures of ingredients in humans must be limited to situations where no irreversible damaging effects are to be expected for the volunteers and where the study goal is reasonably achievable with a study population of limited size.
3. The recruitment of human volunteers should be in line with the "World Medical Doctors Association Declaration of Helsinki" and "the Good Clinical Practice for trials on Medicinal Products in the European Community."
2 Procedure of irritancy assessment
The following text outlines the steps of an assessment of the irritancy of an ingredient or mixture of ingredients. While this text focuses on irritancy, it is understood that other aspects of toxicity have to be considered in parallel before performing tests in humans.
2.1 Initial considerations
Available chemical and physico-chemical data and structure-activity relationships making use of computer programs and databases for the prediction of skin irritation potential should be used.
2.2 Evaluation of irritation
Ingredients or mixtures of ingredients should be tested on animals and humans only at non-corrosive concentrations. This decision may be based on pH and acid/alkaline reserve measurements and on in vitro tests for skin corrosivity. At the present, in vitro methods for the assessment of irritancy have not yet been validated.
2.3 Confirmation by human volunteer testing
On the basis of a low irritation potential as proven by animal or future validated in vitro methods, the skin tolerability of an ingredient or a mixture of ingredient can be confirmed by testing in human volunteers. A number of test protocols are available such as open and closed patch tests, single and repeated-exposure tests. They should be chosen on the basis of the relevant use pattern of the ingredient or mixture of ingredients (1).
While these tests historically have been assessed by clinical methods, non-invasive bioengineering technology such as measurement of transepidermal water loss or of blood flow may provide higher sensitivity and objectivity of these tests and thereby reduce the exposure and risk to volunteers.
However, neither the above confirmatory tests nor the use of bioengineering methods have been validated according to modern scientific criteria. The SCCNFP recommends the Commission to support further research in this area.
2.4 Consumer market surveillance
The evaluation of irritation of an ingredient or mixture of ingredients is not finished with the introduction of respective cosmetic products on the market, but it should continue by making use of data generated by consumer market surveillance and other sources.
3 Ethical considerations
Confirmatory skin tolerance tests of cosmetic ingredients in humans are subject to ethical concerns. In order to take account of these concerns, to minimise the risk to volunteers and to safeguard their rights, test protocols should be submitted to an acknowledged ethical committee and be in compliance with the followings :
i) to supplement non-clinical information,
ii) to confirm that exposure will not cause significant harm, and/or,
iii) in a controlled fashion that minimises subject risk (4).
The investigator(s) in skin tolerability tests of cosmetic ingredients should fulfil the qualifications as mentioned in the CPMP Working Party on Efficacy of Medicinal Products Note for Guidance on Good Clinical Practice (3).
References
(1) Patrick E, Maibach HI: Predictive assays: Animal and man, and in vitro and in vivo. In: Rycroft R.J.G., Menne T., Frosch P.J., (1995) Textbook of Contact Dermatitis. Springer Heidelberg New York.
(2) World Medical Doctors Association Declaration of Helsinki, (1997) JAMA 227: 925-926
(3) CPMP Working Party on Efficacy of Medicinal Products Note for Guidance: Good Clinical Practice for Trials on Medicinal Products in the European Community (1990) CB-55-89-706-EN-C.
(4) Organisation for Economic Co-operation and Development: Development of OECD Test Guidelines for Use in Tests with Human Volunteers. 27 th Joint Meeting of the Chemicals Group and Management Committee, 11 th-13 th February 1998 (ENV/MC/CHEM/RD (98))
ANNEXE 5
SCCNFP/0004/98
Notes of Guidance for testing of Cosmetic Ingredients for their Safety Evaluation
Revision of Annex 7 : Microbiological Quality of the Finished Cosmetic Product
adopted by the plenary session of the SCCNFP of 23 September 1998
1. Preamble.
Skin and mucous membranes are normally protected from microbial attack by a natural mechanical barrier and defence mechanisms. However, protective integuments may be damaged and slight trauma may be caused by the action of some cosmetics that may enhance microbial infection. These situations may be of particular concern when cosmetics are used in the eye area or on mucous membranes or on damaged skin and when used by children under 3 years, elderly people and people showing compromised immune responses. These are the reasons to define two separate categories of cosmetic products in the microbiological quality control limits.
Although a very low number of cases of contamination in cosmetics leading to microbial infections have been reported, it is likely that under-reported clinical microbiological problems (for instance infectious folliculites) associated to the use of contaminated cosmetics are recognised by several dermatologists (to be reported in a separate document). On the other hand microbial contamination may spoil cosmetic products or reduce the intended quality. These statements make it necessary to carry out routine microbiological control of cosmetics, in order to ensure their quality and the safety for customers to use.
2. Categories of cosmetics in microbiological quality control.
In relation with the microbiological quality control, two categories of cosmetics are defined.
Category 1: Products specifically intended for children under 3 years, eye area and
mucous membranes.
Category 2: Other products.
3. Quantitative limits.
The limit for cosmetics classified in Category 1 is: total viable count for aerobic mesophyllic micro-organisms not more than 10 2 cfu/g or ml in 0.5 g or ml of the product.
The limit for cosmetics classified in Category 2 is: total viable count for aerobic mesophyllic micro-organisms not more than 10 3 cfu/g or ml in 0.1 g or ml of the product.
4. Qualitative limits.
Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans are considered the main potential pathogens in cosmetic products. These specified potential pathogens must not be detectable in 0.5 g or ml of the cosmetic product in cosmetics of Category 1 and in 0.1 g or ml in cosmetics of Category 2.
5. Product preservation.
Microbial contaminants have two origins: during production and filling, and during the use of the cosmetic by the customer. From the moment in which the cosmetic unit is opened until the consumer finishes the product, there is a permanent, variable and additive microbial contamination of the cosmetic caused by the domestic environment and the consumer's body (hands and body skin). The reasons for the need of microbial preservation in cosmetics are the following:
5.1. To ensure the microbial safety of cosmetics for customers to use.
5.2. To maintain the quality and specifications intended for the product.
5.3. To confirm hygienic and high-quality handling.
6. The challenge testing
The efficacy of the preservation has to be assessed experimentally during the development process to ensure microbial stability and preservation by challenge testing. Challenge testing is mandatory for all those products that in normal conditions of storage and use, a risk of infection for the consumer or a deterioration of the product exist. The challenge test consists of an artificial contamination of the finished product and a posterior evaluation of the decrease of this contamination to levels ensuring the microbial limits established in products of Category 1 and 2.
The micro-organisms used in the challenge test will be issued from official collection strains from any state in the EU to ensure reproducibility of the test and will be: Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Additional bacteria and fungi might be used for additional specific purposes of the challenge testing. The microcidal activity of preservatives or any other compound in the finished cosmetic must be ruled out in the challenge test by dilution, filtration, neutralisers or any other means. The experimental performance of the microbial controls and the challenge tests must be laid down and validated by a microbiologist.
7. Good Manufacturing Practice.
In order to accomplish with the Good Manufacturing Practices and Microbial Quality Management, manufacturers of cosmetics have to define and follow specific cleaning, sanitation and control procedures to keep appropriately clean and free of micro-organisms that could be harmful for the consumers or adverse for the quality of the cosmetics. These proceedings will include procedures to microbiology control raw materials, bulk and finished products, packaging components, personnel, equipment and locals.
