Re-Evaluation of Animal Numbers and Costs for In Vivo Tests to Accomplish REACH Legislation Requirements for Chemicals – a Report by the Transatlantic Think Tank for Toxicology ( t 4 )

ReACH (Registration, evaluation, Authorization and restriction of CHemicals), the new eU chemicals regulation, is the largest ever investment into the safety of chemicals and consumer products. Acknowledging the fact that we lack about 86% of the safety testing data for existing chemicals (White Paper, 2001, Roe et al. 1997), the eU legislation from 2006 aims to collect such data for all chemicals produced or marketed in quantities of more than one ton per year (ReACH, 2006). the legislation lists the specific information requirements in its annexes for a given chemical depending on the overall production/marketing volume per year. During the political decision process, several attempts were made between 2001 and 2005 to estimate the costs of ReACH, both financially (see for example Pedersen at al., 2003, RPA and Statistics Sweden, 2002) and with regard to animal numbers required (IeH Report, 2001, van der Jagt et al., 2004, Höfer et al., 2004). Since then, no such effort has been undertaken. Several sources of information now allow the estimates to be reassessed: 1) The legislation has been finalized, changing some testing requirements (ReACH, 2006). 2) the eU has been enlarged and now includes 27 instead of 12 countries (3 countries on 1st January 1995, 10 on 1st May 2004 and 2 more on 1st January 2007) compared to the data from before 1994 used in earlier studies. 3) Progress has been made with regard to the availability of alternative methods. 4) testing guidance on information requirements has been developed for industry (eCHA, 2008a-c, Regulation 440/2008). 5) the preregistration of substances by industry ended in December 2008.


Introduction
ReACH (Registration, evaluation, Authorization and restriction of CHemicals), the new eU chemicals regulation, is the largest ever investment into the safety of chemicals and consumer products.Acknowledging the fact that we lack about 86% of the safety testing data for existing chemicals (White Paper, 2001, Roe et al. 1997), the eU legislation from 2006 aims to collect such data for all chemicals produced or marketed in quantities of more than one ton per year (ReACH, 2006).the legislation lists the specific information requirements in its annexes for a given chemical depending on the overall production/marketing volume per year.During the political decision process, several attempts were made between 2001 and 2005 to estimate the costs of ReACH, both financially (see for example Pedersen at al., 2003, RPA andStatistics Sweden, 2002) and with regard to animal numbers re-quired (IeH Report, 2001, van der Jagt et al., 2004, Höfer et al., 2004).Since then, no such effort has been undertaken.Several sources of information now allow the estimates to be reassessed: 1) The legislation has been finalized, changing some testing requirements (ReACH, 2006).2) the eU has been enlarged and now includes 27 instead of 12 countries (3 countries on 1 st January 1995, 10 on 1 st May 2004 and 2 more on 1 st January 2007) compared to the data from before 1994 used in earlier studies.
3) Progress has been made with regard to the availability of alternative methods.4) testing guidance on information requirements has been developed for industry (eCHA, 2008a-c, Regulation 440/2008).5) the preregistration of substances by industry ended in December 2008.enforcement of ReACH began in June 2007, with the setting of deadlines for the registration of substances (tab.1).ReACH also differentiates between phase-in and non phase-in substances."Non phase-in" refers to all substances that have been notified according to Dangerous Substances Directive 67/548/eeC and to those newly introduced to the market after the implementation of ReACH.It is often overlooked that ReACH, with its new testing requirements, is also applied to all new chemicals to be notified.Phase-in substances are all substances that were on the eC market before 18 September 1981 ("existing chemicals") and those previously designated as polymer and no longer considered as such.these three categories, excluding newly introduced chemicals, are classified in the eSIS (european Chemical Substances Information System) as ELINCS (European List of Notified Chemical Substances), eINeCS (european Inventory of existing Commercial Chemical Substances), and NlP (No longer Polymer), respectively.
ReACH has planned a registration deadline for substances according to the correspondent circulating volume (tab.1).6) Other high production volume chemicals safety programs have advanced, giving indications of already existing data.taking these information sources into account, a re-evaluation of estimates was carried out for in vivo test requirements.

Considerations from preregistration of substances
ReACH requires the registration of individual chemical substances, those combined in preparations, and those intentionally released from articles.Registrations are submitted to the european Chemicals Agency (eCHA) and apply to substances manufactured or imported to the eU in annual quantities of one ton or more.However, substances related to medicinal products for human or veterinary use, food or feedstuff, and a few other specific cases -such as elemental substances (O 2 , N 2 , etc.) and substances occurring in nature if they are not chemically modified -are exempted from registration (Article 2, Annexes IV and V). this tool to obtain business information.However, this element alone cannot explain the great number of preregistrations received.
It is noteworthy that the 2008 preregistrations are much closer to an earlier estimate made by RPA and Statistics (RPA and Statistics Sweden, 2002).RPA and Statistics had performed a survey of industry, competent authorities, and associations through a questionnaire about manufactured, imported, or used chemical substances.this questionnaire was able to record eINeCS chemicals, plus nearly 30,000 new chemicals placed on the market annually as well as about 100,000 intermediates isolated annually in the EU.Intermediates can be classified in eSIS but without information about quantity.Most uncertainty in the RPA and Statistics report concerned the unknown amount of each chemical produced.About 23% of intermediates that are effectively placed on the market generate some degree of double counting.For these reasons, the RPA report assumes the possibility of four scenarios as depicted in tab, 3. Numbers depicted in Scenarios 3 and 4 are now closer to the real number of chemicals that have been preregistered.the previous underestimation of substances preregistered has several reasons: 1) the Pedersen et al. (2003) report is based on IUClID, which contains information on substances manufactured in the eU in volumes ≥10 tons/year in the period 1991-1994.
the preregistration phase is now over and the preparation of a registration dossier should be ongoing for each preregistered substance.
On 18 December 2008 (eCHA press release, 2008) eCHA published the first list of preregistered substances in accordance with the preregistration date.Some 65,000 companies have made as many as 2,750,000 preregistrations, far exceeding the 180,000 preregistrations from 27,000 companies originally expected (eCHA Memo, 2008).By suggesting registration deadlines, companies indicate the likely trade and production volume of their chemicals.eCHA has further screened all these entries by combining compounds with the same eC number or CAS number and by deleting some articles.the latest published list (eCHA Press Release, 2009) now contains 143,835 substances.Among those, 54,686 have a 2010 deadline for registration, 59,599 a 2013 deadline, and 29,550 a 2018 deadline.these numbers are quite different from those foreseen in previous assessments for ReACH implementation.In particular, most of the work done to prepare for ReACH was based on a report written by Pedersen et al. (2003).their estimation (tab. 2) was based on data reported in the IUClID (International Uniform Chemical Information Database), which contains information on substances manufactured in the eU in the years 1991-1994.Even if no official numbers are available it appears that a large abuse of preregistration occurred from contract labs, consultants, and other companies who intended to exploit 2) the report includes no substances which are present in IU-CLID but classified as NLP (No Longer Polymer).Eventually, NlP also have to be preregistered.Within the scheme of the notification of new substances (Directive 67/548/EEC) polymers are subject to special rules.the term polymer was further defined in the 7th Amendment of the Directive 67/548/ eeC (Directive 92/32/eeC, 1992).this change means that some substances which were considered to be polymers under the reporting rules when the european Inventory of existing Commercial Chemical Substances (eINeCS) was established are no longer considered to be polymers under the 7 th Amendment.NlP substances require the same preregistration as eINeCS substances.this adds about 700 substances to the preregistration, even though this number is still an underestimation since notification of NLPs was on a voluntary basis.3) Both reports were made before the official publication of ReACH based on draft versions of the regulation.For example, none of them include released substances from articles, even though released substances will reduce the possibility of exposure based waiving rather than increasing the overall number of circulating chemicals.4) Both reports were based on information collected within the borders of the eU at a time when it was composed of only 12 Member States before 1995.At the moment of ReACH implementation, the eU included 27 Member States and the regulation is now also enforced in the eeA (european economic Area), i.e. in three more countries.At the moment, ReACH has been therefore implemented in 30 countries.5) According to the european Chemical Industry Council (CeF-IC, 2009), during the years 2002-2007, chemicals sales grew slightly more slowly than consumption (4.5% versus 5.2%).
The average growth rate of imports over the last five years (7.0%) considerably exceeded that of exports (3.6%).A growth rate of about 5% annually translates to 97% growth from 1994 to preregistration in 2008, based on the IUClID data.

Analyses of preregistered substances
the list provided by eCHA includes, for each preregistered substance, the eC number, the CAS number, the chemical name, and the earliest registration date.this allows a more detailed analysis so we can determine whether all of the listed substances will be fully registered and how many tests will be required to fill the Chemical Safety Report (CSR), as required in Annexes VII-x. the initial digit of the eC number gives indication about whether the chemical is in the eINeCS, the elINCS, or NlP lists.eINeCS contains all substances that were reported to be on the market in europe between 1 January 1971 and 18 September 1981.The total number of substances classified in eINeCS is 100,204.All substances placed on the eU market after 18 September 1981 are classified as "new" chemicals and listed in the elINCS database.the elINCS database includes From the eC number, it is possible to identify to which category a substance belongs, since all eINeCS chemicals start with "2," all elINCS chemicals start with "4," and all NlP chemicals start with "5."Following these assumptions, the preregistered substances are divided and sorted as listed in tab. 4. Tab. 4 gives the numbers for all substances previously classified in the eSIS database that have been preregistered, plus some others that have no eC number and thus were not considered in the previous evaluations.
Considering eINeCS and NlP as Phase-in substances, ELINCS as non phase-in substances, and unclassified substances as "others," the preregistered substances can be further grouped (tab.5).
Most elINCS substances have been preregistered (3,827 nonphase in substances vs. 4381 substances in elINCS).the high number of preregistrations in this class can be easily explained as a precautionary measure adopted mainly by downstream users handling those chemicals.Non-phase-in substances will need extra assessment only in the case of a new use resulting in a new exposure scenario requiring evaluation.Reasonably, this is the case only for a limited number of substances.However, if production has increased since original notification within the last 27 years, additional information requirements might also emerge.this might hold especially true for new chemicals that are now suggested for the 2010 deadline, which means the notifier is expected to have larger production volumes or specific concerns, triggering testing demands typically not met for new chemicals.
Phase-in substances are exactly the same as the number of substances present in the eINeCS and the NlP, with only one NlP missing.It's surprising that none of them is in the lower tonnage level that has a 2018 deadline.
"Other" substances are those that had no eC number during preregistration and now have been given a new list number in the eC format.In order to make registration easier, eCHA assigned new eC numbers starting with "6" to substances without previous eC number but with a CAS number (6-eC class), and starting with "9" to substances that previously had neither an eC number nor a CAS number (9-eC class).the numbers of these substances are listed in tab.6.
the presence of 22,015 substances with no previous classification (neither EC number nor CAS number) is astonishing, even more so because 4,333 of those seem to be on the eU market at a tonnage level higher than 1000 tons/year, which means a 2010 registration deadline.A representative sample of such compounds has been analysed below.

Qualitative assessment
Following the assessment of the number of substances that have been preregistered in ReACH, some more qualitative considerations are required about what exactly is present in this list.
As stated in REACH definitions (Article 3), a substance is "a chemical element and its compounds in the natural state or obtained by any manufacturing process, including any additive necessary to preserve its stability and any impurity deriving from the process used".According to this definition, the same chemical entity can be included in multiple registrations, possibly explaining the high number of preregistered substances.
Certainly, various individuals and companies abused the preregistration tool put into effect, since preregistration gives the right to participate into the forum responsible for the registration of that substance.these include Contract Research Organisations (CROs) that want to propose new tests for the substance, consultants who want to be involved in the registration process, Among the substances within the eN 9-class there are 13,692 entries with the substance name starting with "reaction mass."On the other hand, the query in the list of the field "reaction product" returns 4,468 entries.these entities are considered as Substances of Unknown or Variable Composition, Complex Reaction products and Biological materials (UVCB).UVCBs require that components as high as 10% are identified and characterized.Moreover, the source of the material should be very well described.It can be that this data is already present in the full record, which is accessible only to eCHA and the registrants.

Further considerations on number of preregistrations
As listed in tab.5, the 100,906 substances from eINeCS plus 39,102 non-classified substances results in a total of 140,008 substances that may require extensive testing for registration.this number is substantially larger than the previously foreseen number of 29,342 and understanding the reason for this enormous discrepancy is of utmost importance.
ReACH originated from the white paper "Strategies for a future Chemicals Policy," presented in 2001 (White paper, 2001).This first document mentioned only chemicals in the eINeCS and elINCS lists.today, ReACH includes isolated intermediates, chemicals in any preparations, as well as chemicals released from articles.Previous assessments foreseeing the impact of ReACH on the eU economy were based on the original White Paper and on other draft versions that were in general very similar.The final regulation was published on 30 th December 2006.
this is probably why earlier estimates foresaw only 29,342 substances for registration ( tab. 2).All NlP must be immediately added.there are only 703, increasing the estimate only slightly, to approximately 30,000 substances.
In addition to the evaluation of 30,000 substances, isolated intermediates can be very important, even though the exact type competitors who want to acquire information about the substance, and many others.
However, the main impact on the number of preregistrations is attributable to mistakes made by registrants.In fact, there is a high level of fear that ReACH can close the commercial activity of companies that are not compliant with the new regulation.In the eU most companies of chemical industry are small and medium-sized (SMe); most of them only deal with preparations and articles and lack specific chemical toxicology competence.At the moment there are no official data, but it is well known that many downstream users have registered important substances they are using, even if purchased in europe.Another indication of misunderstanding is the presence of all substances that are already classified in the ELINCS database when it was clearly stated that non phase-in substances (i.e.substances already present in the elINCS database) did not require preregistration.
On its website eCHA also considers possible reasons for mistakes in 14,000 entries, including difficulties with English (the only language admitted for preregistration) and the classification of mixtures (eCHA Press release, 2009).eCHA has already skimmed some of the initial registrations (eCHA Press Release, 2008).Nevertheless, eCHA has no authority to automatically delete a registration without properly communicating with the registrant.
In order to better understand the false entries, some of them have been randomly selected from the 39,102 substances that have no eC number.Reasoning about each single entry is difficult.Only legal entities that have preregistered have full access to the eCHA database, which may contain further explanations.were not yet counted and that some of the countries were not part of ReACH, the 18% increase of the chemical business seems to be realistic.

Conclusion on number of expected full registrations
Current information is inadequate to provide a precise account of the number of substances that will require further in vivo testing for preparation of the registration dossier.However, an approximation can be attempted.
All substances that have elINCS number have been notified and, in general, they already have what is needed to fulfill ReACH requirements.that is why they are not counted for this study, even though some may require more testing.Substances belonging to the "6" and "9" eC categories are also excluded, because our samples showed that most of them are either out of the scope of ReACH or already preregistered with another name and proper eN number.Of course, the elimination of both elINCS and 6/9 eC classes from the category of chemicals requiring further in vivo testing is not fully correct.However, these omissions should be counterbalanced by the number of substances considered in this study that will either quit registration or will be registered in a lower tonnage level.the omission of all substances not previously classified in ESIS surely leads to a large underestimation of the total number of registrations that will be required.
All preregistered phase-in substances, i.e. substances classified in EINECS and NLP database, are included in the estimate, assuming that companies know their trade and production volumes.this total of 100,906 substances will be split as: • 47,858 present in the eU in quantity higher than 1000 tons/per year and under the provision of ANNex x of ReACH • 53,048 present in the eU in quantity higher than 100 tons/per year and under the provision of ANNex Ix of ReACH this can be considered a quite realistic scenario for the number of chemicals that need full registration with CSA/CSR.
Another approach is to start from the hypothetical number of chemicals in the Pedersen et al. (2003) estimation, which is based on a realistic picture of the situation in the eU for the years 1991-1994.these numbers are increased by a factor derived from the growth of the chemical industry as depicted by CeFIC (2009), i.e. by a factor of 1.97 due to the general increase of chemical industry plus a factor of 1.18 due to the enlargement of the eU (see above).this approach still represents an underestimation as isolated intermediates and released substances from articles are not taken into consideration.UVCBs are substantially excluded in all these calculations.Presumably, most of them are mixtures of other known chemicals and a maximum exploitation of read-across and grouping opportunity is expected.
All these assumptions are summarized in tab. 7. Presumably, the real situation will lie between scenarios 2 and 3 of this table, with a total number of chemical to be registered within 68-100,000.and amount are recorded nowhere.RPA and statistics (2002), in its survey, counted as many as 85,000 possible intermediates that need to be registered.the addition of this contribution adds up to approximately 115,000 substances, still far from the final number of preregistered substances.More inputs should be found elsewhere.even if preregistration of isolated intermediates is mandatory, when they are not marketed they are exempted from the preparation of a dossier and, where strictly controlled conditions of use apply, they have very reduced data needs.the number of intermediate chemicals in ReACH is difficult to estimate but, especially at higher tonnages, this will apply to many basic chemicals used to synthesize more specialized products.
Phase-in substances are considered by ReACH as the chemical substances circulating in the eC before 1981 and registered in IUClID.that is why previous estimations consider the substances that were present in IUClID in the period 1991-1994.even though this estimate is correct from a qualitative point of view, since eINeCS contains today the same number of chemicals, from a quantitative point of view it completely underestimates the quantity of each chemical currently circulating in the eU.In theory, the picture recorded by IUClID in years 1991-1994 should be consistent with today, since new chemicals should all have been recorded in elINCS.However, it does not consider the discrepancy between the increasing rate of chemical industry growth and the eU enlargements that occurred in 1995 with the accession of Austria, Sweden, and Finland to the eU, the 2004 accession of Czech Republic, estonia, Hungary, latvia, lithuania, Poland, Slovakia, Slovenia, Malta, and Cyprus, and the latest enlargement in 2007, which included the accession of Bulgaria and Romania.Moreover, ReACH is now also enforced in the countries of the european economic Area (eeA), which incorporates Norway, liechtenstein, and Iceland.
The precise quantification of the contribution from these countries is very difficult as source information is hardly accessible.However, it is well known (Angerer et al., 2007, CeFIC, 2009) that the chemical industry in some of the new member states plays an important role in the economy.Commercial activities in most of these countries were mainly with Russia and eastern countries, rather than eU-15; i.e. they contributed little to the eU-12 chemical commercial business in 1991.Moreover, most of the production was focused on "basic chemicals" rather than "specialty chemicals."the difference between the two is in both volume and function, as specialty chemicals are manufactured in lower volumes and used for specific purposes.It is reasonable to assume that basic chemicals are all present in eINeCS, leading to a much higher volume of those chemicals circulating in the eU today.
last, but not least, the average annual growth rate of the chemical industry was much higher in most of the new member states (e.g.Poland, Czech Republic, estonia) than in the old member states (Angerer et al., 2007, CeFIC, 2009).According to CeFIC (2009) in 2007, eU-15 sales in the chemical sector represent 501 billion euro versus 94.5 billion euro in the rest of europe.Considering that in 1993 Austria, Finland, and Swe-study is waived when a full study is required.A developmental toxicity study is performed on two species and an expert decision is based on the outcome of the first test and all other relevant available data.In vivo skin irritation is still written in the Annex although it is no longer obligatory as an in vitro alternative is now fully accepted.
Actually, ReACH requires many more tests, such as those for the characterization of physicochemical properties, in vitro tests, and in vivo tests on plants and invertebrates.All these tests remain out of the scope of the present study.ReACH also refers to hazards such as respiratory sensitization, endocrine disruption, and neurodevelopmental toxicology, but these are not reflected in the Annexes and therefore are also not considered.
If all preregistered substances required all in vivo testing, the final costs and the number of sacrificed animals would be enormous.the real number of tests will be the result of several factors, including:  the general rules for adaptation of the standard testing regime as set out in Annexes VII to x.
For each of these factors, Pedersen et al. (2003) have tried to estimate the weight in the final count.In the present study, the same approach is applied to the number of preregistered substances that will probably undergo a full registration process, i.e. 47,858 substances under Annex x and 53,048 substances under Annex Ix provisions.However, new testing strategies derived from the guidance to industry for endpoint specific information requirements and published by eCHA (2008a-c), offer a re-analysis of the available data and the state of the art of in silico and in vitro alternatives, including novel reduction and refinement alternatives.Precise quantification of these factors is not possible at the present level of implementation of ReACH because of the lack of knowledge concerning the acceptance of alternative approaches.tab. 9 reports the percentage values of available data. the percentage values include both existing data and other informa-• existing, available information: Some of the substances have already been tested and/or covered by other legislation that provides for an equivalent level of protection (e.g.Biocidal Products Directive, Medicinal Products Directive, US High Production Volume chemical program).In ReACH there is the obligation to make use of any available information and share any available result if obtained from an in vivo test.

Existing and promised data
• Waiving: Sometimes the applicability of a test depends on the physicochemical properties of the substance.Some other tests are triggered by the response of another test.
• exposure based waiving: Some of the testing for the higher annexes may be omitted based on a comprehensive exposure assessment developed in the Chemical Safety Report.Provisions regarding this opportunity have been recently updated (Regulation 134, 2009).
• (Q)SAR (Quantitative Structure Activity Relationship), group- ing, read across: A theoretical model can predict the property of a substance (QSAR, in silico approach, see also article by Hartung and Hoffmann in this issue of Altex), or a group of substances belonging to the same chemical class can be grouped and only one of them is tested (grouping), or a property of a substance can be derived by the existing information for a similar substance (read across).
• In vitro methods and other alternative approaches are accepted when suitable, as defined in Annex XI of REACH.contrast, substances under Annex x provisions always require a full developmental study and the screening test is waived.the possibility of a waiver is inherent to the physicochemical characteristic of the substance and therefore is independent of the tonnage that needs to be registered.the values proposed by Pedersen et al. (2003) are summarized in tab.10.Unfortunately, at the moment there are no data to confirm these numbers.there are other minor cases providing possibility for waiving.For example, a metabolite is covered by the testing of the starting substances.Since these opportunities have a minor impact on the final numbers, they are not taken into account in the final calculation.Moreover it should be noted that screening studies concerning safety factors for higher tonnage bands are not allowed, providing a substantial increase in testing demand for substances cover by Annexes Ix and x provisions.

Final number of tests required for registration: current guidance and accepted methods
tab. 11 is the result of the combination of both tables 9 and 10 applied to 53,048 and 47,858 substances to be registered according to the provisions in Annex Ix and x respectively.the percentage of existing data is relative to the number of required tests, i.e. on the total number of substances subtracted by the percentage that can be waived.to estimate the total number of test animals needed for each endpoint, van der Jagt et al. ( 2004) have compiled information provided by some referenced organizations with extensive experience in this field.The final figure is the result of the application of official guidelines, as described in the Council Regulation 440/2008, which essentially implements the correspondent OeCD guidelines as properly listed by tion available to Industry for sharing (the so-called "promised data").Pedersen et al. (2003) also consider the contribution of voluntary initiative on providing data, based on the information available in the US on the HPV Challenge Program, which covers base set tests for higher production volume substances.Regarding the low volume substances, there is an investigation performed on substances that are manufactured in Germany which are classified in IUCLID.Since the values shown in Tab. 10 are the results of a general survey, it is assumed that they also include data that are already present in IUClID and in the US HPV Program.
the US HPV Challenge Program "challenges" companies to make publicly available data on chemicals produced or imported into the United States in quantities of one million pounds or more per year (about 500 tons).Companies have sponsored more than 2,200 HPV chemicals, with approximately 1,400 chemicals sponsored directly through the HPV Challenge Program and over 860 chemicals sponsored indirectly through international efforts.Only directly sponsored HPV Challenge Program chemicals make it to HPVIS (High Production Volume Information System).Currently, the HPVIS database contains over 340 submissions, representing almost 900 chemical substances (ePA, 2009a).earlier (Bremer et al., 2007) we evaluated the most critical part with regard to animal numbers and costs, i.e. reproductive toxicity, and available test results.the HPV database contains eight one-generation and 10 two-generation studies as well as 43 developmental screening studies (for comparison, the eU New Chemicals Database includes 55 one-and 14 two-generation studies and no screening studies for 4,400 registered new chemicals).These figures for available data are ranging from 0.3-2%-substantially lower than assumed 7-22%.At the same time, it is highly unlikely that many further reproductive toxicity studies will be available for chemicals by individual companies and not used for registration purposes.
Another approach made by Allanou et al. (1999) counted the number of entries in the eU HPV list extracted from IUClID management software.From 2,465 substances, Allanou at al. checked whether: i) the field of the toxicological endpoint was filled in the correspondent entry and ii) whether this string was a number.However, also assuming that all these 2,465 substances have been preregistered in the category of substances with a deadline of 2010, the percentage that came out from this query is less than 1% and it would have a negligible impact to the final results (tab.9).

Waiving
In Annexes VII through x, ReACH regulation gives some standard rules for each endpoint about how to perform the test in the event that no data are available.ReACH also provides rules indicating when a test may be waived: if there is no concern regarding exposure in a scientific demonstration, if performing the test is technically impossible, or when the result of a test is clearly derived from the information obtained for another endpoint.For example, a full developmental toxicity study (ReACH § 8.7.2) shall only be performed for a substance under Annex Ix provisions when there is a positive response from a developmental toxicity screening (ReACH § 8.7.1).In ) and read across.the basis of (Q)SAR approach is the hypothesis that a chosen property of a substance can be described in relation to the similarity of its chemical structure, as a whole or partially, with other chemicals for which the same property is known (Benfenati, 2007).the capability of (Q)SAR is evolving rapidly, aided by expanded computational technology and increased availability of databases providing more and more information on chemicals.even though the capability of (Q)SAR modeling is clear, it is true that (Q)SAR tool can rarely be used as a standalone technique for the evaluation of complex toxicological endpoints.As described in the eCHA (2008a-c) technical Guidance Documents, (Q)SAR is well placed in the ItS (Integrated testing Strategy) for the assessment of substance toxicological properties, and its main role is to point out specific properties and to optimize the number of tests and animals used for each endpoint.According to van der Jagt et al. ( 2004) the extent to which (Q)SAR and read across have been used in the US HPV Challenge Program Höfer et al. (2004).For the present study, the average number of animals per test as evaluated by van der Jagt has been considered for all endpoints except: ReACH § 8.7.1.developmental toxicity screening, ReACH § 8.7.2 developmental toxicity study (two species), and ReACH § 8.7.3 two generation reprotoxicity.In fact, van der Jagt et al. ( 2004) did not take into account offspring, which are also test animals included in eU animal use statistics.For these endpoints, the number of animals per test as proposed by Höfer et al. (2004) was considered.
Tab. 12 lists the final result of this approach, showing that implementing ReACH according to current guidance would require 141 million vertebrate animals!this value is clearly unacceptable, for both ethical and financial reasons (Bottini and Hartung, 2009).Feasibility should be taken into account as well, since europe lacks the resources to run so many tests in such a short period of time (IeH Report, 2001).However, the introduction of Annexes VII through x clearly states that: "Before new tests are carried out to determine the properties listed in this Annex, all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) shall be assessed first."In this sense, REACH is one of the first legislative acts introducing a spirit of change in the field of toxicology, even though their full acceptance will require some time (Hartung, 2009).

Tab. 11: Total number of tests required for each endpoint based on actual preregistration considering both estimated existing data and possible waiving
This calculation of number of OECD guideline tests on individual chemicals is based on scenario 2 (table 7), i.e. the actually preregistered substances corrected for likely mistakes.It considers both existing data (tables 9) and possible waiving ( the application of in vitro methods, which are also strongly supported by the ReACH regulation, should help lower the number of animals required.Unfortunately, the development of such approaches for the complex and animal demanding endpoints is not even close to either validation or to regulatory acceptance.
In the following paragraphs all endpoints requiring in vivo testing are considered.In each case, some guesswork is used in an exercise to simulate the application of the different OeCD tGs and to calculate the number of animals required in each step.Most of the assumptions are derived from personal experience.these assumptions were checked for plausibility by six ReACH experts from industry and a regulatory authority.even though there are no statistics supporting this conjecture, it may give a picture of the scenario required for ReACH compliance.
the average number of animals per tested substance is derived according to the corresponding guideline.lower numbers can result by testing several chemicals with one control; higher numbers might be necessary if the concentration response is not conclusive.Moreover, it should be considered that some of the most complex tests, such as 90 day repeated dose toxicity, carcinogenicity, etc., can be performed at the limit dose only instead of the three dose levels when there is no sign of toxicity for the chemical.limit dose testing, where foreseen in the test guideline, can save up to 50% of the animals required for testing. is up to 44% for human health endpoints and 35% for environmental effects.Pedersen et al. (2003) consider the percentage of acceptability of (Q)SAR results for each endpoint.tab.13 lists the results obtained by applying RPA and Statistics approach to the extrapolated total number of tests.On average, the "good" (Q)SAR approach implies a reduction of 4.6%, the "fair" approach a reduction of 1.9%, and the "poor" approach a reduction of 0.1%.If this is true, the (Q)SAR benefit is practically negligible.the main hindrance in the applicability of (Q)SAR models is the limited applicability domain, which is generally well explained and measured for each model and based on the similarity of the structure of interest with the substances that have been used to build up the model.
Moreover, it should be further highlighted that (Q)SAR calculations are based on specific organic chemicals with a well defined structure.For this reason, any inorganic compounds, organometallic compounds, mixtures, and UVCBs are excluded by default.Barrat et al. (2007) tested 400 chemicals randomly selected from eSIS HPV and lPV. the output was that (Q)SAR could be taken into consideration for only half of them, just by excluding inorganic and ionic compounds, complex mixtures, and those chemicals with no unique chemical structure.
Notifiers can apply read across to categories of chemical structures, especially in the high tonnage area where there are a lot of refinery/production streams of broadly similar material.

Tab. 12: Total number of animals required for each endpoint based on actual preregistration considering existing information and waiving but no alternative methods
This calculation of number of animals for OECD guideline tests on individual chemicals (table 12) is based on scenario 2 (table 7), i.e. the actually preregistered substances corrected for likely mistakes.It considers both existing data (tables 9) and possible waiving (table 10) but no alternative approaches.Animal numbers are from Höfer et al. 2004.marketed at concentration higher than 10t/y, 100t/y, and 1000t/y respectively.the estimated costs for running OeCD tG 405 is about 1,100 euro per tested chemical.

Skin sensitization
the OeCD tG 429 (local lymph Node Assay, llNA) is the standard requirement from Annex VII onward.However, OeCD tG 406 using 15-30 guinea pigs (maximization test vs.Buehler) can also be used according to the guidance (eCHA, 2008).eCB assumes 20% existing data.No testing is required for skin corrosives (3%).Since no validated alternative approaches exist and sensitization cannot be established by read-across, 77% of substances will require testing.Actually, some alternative approaches are under development.(Q)SAR activity in this field is quite interesting and several models are now publicly available.General perception is that (Q)SARs can be effectively employed as part of a battery of methods together with results from in vitro testing, as demonstrated by the results of two eU integrated projects, Caesar and Sensit-iv (www.caesar-project.eu;www.sens-it-iv.eu).In the near future, they may provide tools for the reduction of the number of animals used for skin sensitisation assessment.At the moment, there is no general acceptance and/or knowledge and therefore no contribution from alternative methods is considered in the present study.No alternative method is considered suitable in the eCHA technical Guidance (eCHA, 2008a).
Among the chemicals that will be tested for skin sensitisation in vivo, it is assumed that two-thirds will apply the llNA as the clearly preferred test in the legislation and one-third will use guinea pig methods (23 animals assuming equal use of GPMt and Buehler).It is also assumed that in half of the cases, the validated reduced llNA is used, consuming only eight animals instead of 16.Final counting assumes the application of each guideline for 25.7% of all registrations.Cost estimation is about 3,290 eUR for llNA and 2,000 eUR for the reduced method.A study according to OeCD tG 406 can cost up to 4,000 eUR.

Acute toxicity
Oral route OeCD tG 420, OeCD tG 423 or OeCD tG 425 (accepted refinement and reduction methods) are required from Annex VII onward with a second route OeCD tG 403 (inhalation) or OeCD tG 402 (dermal) from Annex VIII onward.eCB assumes 56% oral and 34% of other routes available.It is assumed that dermal route is preferred to inhalation by 9:1.3% of skin corrosive substances do not need to be tested.No validated and/or accepted alternatives are available.Notably, tab.7 lists four different scenarios for the number of substances that will need some in vivo testing for registration.Scenario 4 represents roughly the Pedersen et al. (2003) predictions made about five years before the end of the preregistration phase and they are not further considered.Scenario 1 considers a full registration of all preregistered chemicals.this is by far the most pessimistic scenario and it hardly represents the real situation.Scenario 2 is probably quite near to the real situation in terms of total number of registered chemicals.However, the high number of chemicals preregistered at the upper tonnage level seems quite far from the present situation.Predictions in Scenario 3 of table 7 are quite optimistic, but they have a better spread over all 4 Annexes with their requirements and have been chosen for a more detailed assessment of in vivo testing requirements.this approach allows a more detailed distinction between different marketed tonnages that is not possible for the chemicals in the list of preregistered substances.this scenario foresees 44,632 chemicals marketed at level ≥1t/y, 11,570 chemicals marketed at level ≥10t/y, 5,721 chemicals marketed at level ≥100t/y and 6,286 chemicals marketed at level ≥1000t/y.Noteworthy, this corresponds well with estimates given in recent presentations of eCHA representative as to the number of submission expected for end of 2010, which range between 4.000 and 10.000.It is evident that this starting point for the following assumption leads to a large underestimation of the final number of animals necessary for registration and of the related costs.
Costs are either taken from Fleischer ( 2007) survey or, when data were missing, from the personally communicated experience of a private contract research laboratory.

Skin/eye irritation and corrosion
this area has been largely covered by validated and eU/OeCD accepted alternative methods, with the notable exception of eye irritation (OeCD tG 405).eye corrosion is covered, allowing screening for severe irritants first.Testing is required from Annex VIII onwards.
estimates for no testing: • already classified: 73% (ECB) • human data: 1% • skin corrosive: 3% (Hoffmann et al., 2005)  • pH <2 or >11.5: 2% • flammable in contact with air or water: 0.1% • peroxides: 0.1% • acute dermal <400mg: 5% • BfR rule base and (Q)SAR: 10% (Rorije and Hulzebos, 2005)  • In vitro eye corrosive: 5% • Non-validated in vitro: 10% • Read-across: 10%   this means that data are lacking for 26% of chemicals.Of these 45.2% can be gained without animal testing, i.e. 14.3% of chemicals will be tested according to OeCD tG 405.Notably, ongoing validation activities promise to replace this test within few years.Given an average animal use of 1.3 animals per tested chemical, the most optimistic scenario results in the consumption of 10,229, 1,041, and 1,144 animals for chemicals out in 80% of the cases, i.e. for 21.2% of Annex VII chemicals.the false-positive rate for the combination of tests in Annex VIII ranges between 75-95% (Kirkland et al., 2005), which means it is fair to assume that together with the real positives about 90% of chemicals will be considered for in vivo and 80% of these, i.e. 72%, will be carried out.
For Annex Ix and x, the in vivo test shall be considered independent of the in vitro results.the eCB assumed 7% data availability; and assuming again 80% execution of tests, 74.4% of substances will be tested.
the in vivo tests comprise different mutagenicity and the less frequently used germ-cell mutagenicity assays.the guidance document foresees 1-3 in vivo tests, to be chosen among OeCD tG 474, 475, 483, 478 and 486.Requirements for each of these methods are quite different in terms of both animal consumption and costs.the average of 70 used in previous estimates (Höfer et al., 2004) seems realistic, as does the average cost of 10,800 euro per tested chemical.

Carcinogenicity
Animal testing for carcinogenicity is very much discouraged by the guidance: "A carcinogenicity study may, on occasion, be justified.If there are clear suspicions that the substance may be carcinogenic, and available information (from both testing and non-testing data) are not conclusive in this, both in terms of hazard and potency, then the need for a carcinogenicity study should be explored.In particular, such a study may be required for substances with a widespread, dispersive use or for substances producing frequent or long-term human exposures.However, it should be considered only as a last resort." eCB assumes 4% available data, not relevant here.
It is assumed that 1% of the Annex x substances, and 0.1% of suspicious chemicals of the other Annexes will be tested in OeCD tG 451.Application of OeCD tG 453 can be considered as equivalent.
All carcinogenicity studies are quite long and complex, justifying the high cost per chemical of 780,400 euro upon the use of "only" 400 rats per study.

Reproductive Toxicity
Reproductive toxicity evaluation includes the developmental toxicity screening (8.7.1) required in Annex VIII plus developmental toxicity test (8.7.2) and two-generation reproductive toxicity study tiered testing variants for OeCD tG 403 and OeCD tG 402 are under discussion at OeCD. these assumptions are summarized in tab.14, which also includes average costs.
Recently the national Coordinators of the OeCD environment, Health and Safety program have approved a new test guideline on Acute Inhalation toxicity (Draft OeCD tG 436), which uses a significantly reduced number of animals.The final approval of this new guideline is expected in late 2009 (Altex News, 2/2009).

Repeat dose toxicity
A 28-day study in one species is required from Annex VIII onward in the most relevant route.It is assumed that this is 90:9:1 for oral:dermal:inhalation.From Annex Ix onward a 90-day study has to be added.eCB assumes availability of 34% and 7%, respectively.
testing can be waived for no relevant exposure, assumed at 5%, thus resulting in test needs for 61% and 88%, respectively.It is not expected that long-term studies (12+ months, OeCD tG 452) will be triggered.Notably, minimum animal numbers have been assumed with only three dose groups.Additional triggers of testing were not considered.
the use of OeCD tG 452 is discouraged in the guideline (eCHA, 2008) and therefore it has been disregarded from this calculation.Contribution from OeCD tG 453 and 422 is counted with carcinogenicity and reproductive toxicity assessment, respectively.
these assumptions are summarized in tab.15, which also includes average costs.

Mutagenicity
According to the guidance (eCHA, 2008a) Annex VII requires an Ames test, which in case of positive findings shall go to Annex VIII requirements for mutagenicity.Annex VIII foresees two more in vitro tests.According to Kirkland et al. (2005), the Ames test has a specificity for non-carcinogens of 73.9%, which means 26.1% false-positive results.Its sensitivity is about 90%.thus, assuming about 10% real mutagens among chemicals, 23.5% of chemicals would be Ames-positive (9% real and 14.5% false-positive).In case of positive results, typically an in vivo test shall be considered; we assume that it will be carried rats and Fleischer (2007) consider an average price of 328,000 euro for the study on rats.We can assume 2,100 rabbits or 3,200 mice per chemical in case testing a second species is required with a cost of 328,000 euro per test (the lower number of 2,100 was used for the calculation).testing on a second species is specifically recommended in the ECHA guideline.
• OeCD tG 426, adopted on 16 th October 2007: Developmen- tal neurotoxicity.Preferred species is rat and the dosing procedure starts from pregnant females, without specifying how many male rats are used.The final request is for 20 litters, with no indication on the number of parents.We assume that at least 10 males and 10 females are selected as parents per dose group, with three doses per chemical plus one control dose.On average, the DNt study tG 426 requires 1,400 animals and costs 1.1 million euro.
• OeCD tG 415, adopted on 26 th May 1983: One-generation re- production toxicity study (eU Method B.34).Preferred species is rat. the method requires at least 20 females and 20 males per dose, with three doses per chemical plus one control dose.this method is not further considered as it is assumed that the twogeneration study (OeCD tG 416) will be the preferred method of choice until agreement on tG 415 on OeCVD level, even though some proposals for a revision of current strategy are under discussion (Spielmann and Vogel, 2007).
Average litter size of rats is 8.2 (Kidwell and Weeth 1959), while the average litter size for mice, the preferred second species for OeCD tG 416, is 8.3 (Finn, 1963).For rabbits, average litter size is 5.5 (Blasco et al. 1993), resulting in about onethird fewer animals.Notably, while Höfer et al. ( 2004) calculate 3,200 rats for an OeCD tG 416 per chemical, Cooper et al. (2006) estimate only 2,600.For consistency with the previous calculation, the Höfer et al. (2004) figures were used, but about 20% over-estimation in animal numbers for this important component might be possible.
The ECHA (2008a) has published endpoint-specific guidance to industry.In the following, this guidance is translated into an estimate of testing demands: Standard requirements: • Annex VIII (10-100t/y): OeCD tG 421 or 422; since typical- ly 28-day repeat dose study data will be available, we assume required in Annex Ix (8.7.3). the developmental toxicity study is usually tested on one species but testing another non-rodent species is now recommended, basically changing the accepted current procedure for the assessment of reproductive toxicity.there is also a strong interconnection with other endpoints: a positive outcome on reproductive organs from the repeated dose toxicity study can trigger a two-generation toxicity study, while chemicals already classified as carcinogenic do not need reproductive toxicity testing as the risk measures must be adequate.the following animal tests need to be considered: • OeCD tG 421, adopted on 27 th July 1995: Reproduction/de- velopmental toxicity screening test.Preferred species is the rat. the method requires at least 10 females and 10 males per dose, with three doses per chemical plus one control dose.Pups are counted.Höfer et al. (2004) calculate 560 rats and Fleischer (2007) consider an average price of 54,600 euro for the study on rats.
• OeCD tG 422, adopted on 22 nd March 1996: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.Preferred species is the rat. the method requires at least 10 females and 10 males per dose, with three doses per chemical plus one control dose.Pups are counted to give an average of 412 animals per chemical and a cost of 92,000 euro per study.
• OeCD tG 414, adopted on 22 nd January 2001: Prenatal de- velopmental toxicity Study (eU Method B.31).Preferred rodent species is rat, preferred non-rodent species is rabbit.the method requires at least 20 pregnant females per dose, with three doses per chemical plus one control dose.Killing of the females is foreseen one day prior to the expected day of delivery, pups are counted as it is well known that foetuses perceive pain starting from approximately 60 percent of the gestation period (Close et al. 1997).Fleischer (2007) consider an average price of 63,100 euro for the study on rats and 92,500 euro for the study on rabbits.Rabbit consumption is estimated to be 560 per tests while rats are 784 per test.
• OeCD tG 416, adopted on 22 nd January 2001: two-genera- tion reproduction toxicity study (eU Method B.35) Preferred species is rat. the method requires at least 20 females and 20 males per dose, with three doses per chemical plus one control dose.Some of the F1 offspring are mated and F2 offspring are also included in the study.Höfer et al. (2004)  or which have at least examined a relatively broad range of reproductive or developmental parameters (US National library of Medicine's tOxNet-Developmental and Reproductive toxicity study).
The output of the research was about information sufficient for only 71 substances positively classified.Even assuming that 2-to 20-fold more negative study results are available, for the purpose of ReACH this number does not impact on overall test costs and animal use.
Human data are considered even more infrequently and are usually limited to drugs.the US ePA Integrated Risk Information System on high production volume chemicals, which is closest to the ReACH dataset, contained information on reproductive toxicity tests for 17 of about 2,000 chemicals.We will assume 1% of available data for the purpose of this study.
thus, existing data are negligible, and limit the opportunities to carry out read-across or modelling by (Q)SAR.to some extent, grouping of chemicals with testing prototypic members of a series of homologues might be considered, but experience from the drug area has taught that even enantiomers (thalidomide) or slight structural variants (retinoic acid) behave completely different with regard to reproductive hazard.We will assume a saving effect of 5% for the purpose of our study.
the guidance further suggests that substances "for which a mechanism of toxicity has been identified that is causally related to reproductive toxicity … may be reasonably expected to exhibit the same pattern of reproductive toxicity."However, only very few mechanisms of reproductive toxicity are known, which relate to many chemicals, with the possible exception of endocrine disruption.this is not a standard testing requirement in ReACH.thus, grouping cannot play a major role in a decision to waive testing, but we will assume another 5% of waived testing.
Testing can also be waived if the substance is "classified as a genotoxic carcinogen category 1 and mutagen category 3 or carcinogen category 2 and mutagen category 3, or a germ cell mutagen (Mutagen Cat. 1 or Cat.2).For the purpose of our estimate we will assume 5% of such cases.testing can further be waived if "all three criteria … are met": "(a) low toxicological activity (b) negligible systemic absorption and (c) no significant human exposure."Meeting all three criteria is rare and will not exceed 5% of cases.
Data from other animal tests are seen as a critical substitute for reproductive toxicity testing, e.g."sensitivity of repeated-dose toxicity studies for detecting effects on reproductive organs may be less than reproductive toxicity studies because of the lower number of animals per group" and "occur at lower doses during the development of foetuses and young animals than in adults."On the contrary, further reproductive toxicity studies might be triggered "based in a Weight of evidence assessment." Notably, "Repeated-dose toxicity studies may also provide indications to evaluate the need to investigate developmental neurotoxicity endpoints."this is further explained as "Relevant triggers could be if the substance has been shown to: 1) Cause structural abnormalities of the central nervous system 2) Cause clear signs of behavioural or functional adverse effects 67% use of OeCD tG 421 and 22% use of OeCD tG 422, but "dependent on the nature of the alert(s)…it may be more appropriate to conduct a two-generation reproduction study … or a prenatal developmental toxicity study" in addition; for this estimate it will be assumed that this is the case for 1% of the chemicals each for OeCD tG 414 and OeCD tG 416.testing in a second species is normally not considered.
• Annex Ix (100-1000 t/y): required method is OeCD tG 414, occasionally including a non-rodent species ("a study in a second species will normally be required when the first study is negative," for the purpose of this estimate we will assume that this is the case for 80%); in case of special concern, developmental neurotoxicity (DNt) testing should be taken into consideration as well.
• Annex X (≥1000t/y): required method is OECD TG 416, oc- casionally including a non-rodent species; OeCD tG 421/422 and OeCD tG 414 are not required; in case of special concern, developmental neurotoxicity (DNt) testing should be taken into consideration as well.
Upgraded testing requirements: "Human exposures, particularly for consumers, are close to the levels at which toxicity might be expected," we will assume that this is true, and in 1% of the cases the substance will move one level up.
Notably, "clear evidence of adverse effects" from OeCD tG 421 or 422 will result in "no requirements for the conduct of a two-generation study at higher tonnage levels.""A negative result … will provide reassurance of the absence of this hazardous property."For the purpose of our calculation, since reproductive hazard is rare and these screening tests typically do not give "clear evidence" we will assume 5% of such waiving for Annex Ix and no effect for Annex x, since here no OeCD tG 421/422 testing is foreseen.Noteworthy, in the european Commission regulation 134/2009 amending Annex xI of ReACH (published in the Official Journal of the European Union on 16 February 2009) it is explicitly stated that derived no-effect levels "from screening test for reproductive/developmental toxicity shall not be considered appropriate to omit a prenatal developmental toxicity study or a two-generation reproductive toxicity study" prohibiting this major waiving opportunity.
existing information allowing reproductive toxicity testing to be waived is rare (about 100-200 substances are currently recognized as reproductive toxicants).
An extensive survey (Bremer et al., 2007) has been developed over: • • Studies conducted according to recognized test guidelines one-generation reproductive toxicity test (Bremer et al., 2006, Spielmann and Vogel, 2007, eCetOC, 2008).However, in vitro positive results are considered a trigger for further testing ("a positive result in a validated in vitro test could provide a justification for further testing").taking these considerations together, maximally 21% of animal tests can be waived based on 1% existing data, <5% grouping, <5% endocrine disrupters, < 5% classified carcinogens and germ cell mutagens, and <5% negligible exposure.A contribution of in vitro or (Q)SAR alternatives is not foreseen.Noteworthy, there is some saving potential applying only a limit test with one dose >1000mg/kg and control group in all test guidelines, but the ReACH guidance to industry does not even mention this opportunity, which was thus not further considered.All these considerations are summarized in tab.16, which also lists average costs.

Fish bioaccumulation
The official test (usually OECD TG 305) is required in Annex Ix as well as for PBt substances.
the guidance is cautious (eCHA, 2008b): "In view of the importance of this endpoint in the assessment of a chemical, and the relatively small number of substances that have been properly tested, a cautious approach is needed."eCB assumes data availability at 19%. the applicability of (Q)SAR models for elucidation of bioaccumulation is quite extensive for this endpoint (Weisbrod et al., 2007, Caesar Project 2009), providing further contribution to lower the demand of in vivo testing.
It is assumed that 10% of chemicals will actually be tested from Annex Ix and x.Furthermore, it is assumed that about 1% of substances from Annex VII and VIII might qualify as PBt.

Short-term and long-term fish acute toxicity
Short-term toxicity testing on fish is required in Annex VIII, i.e., for substances marketed at a tonnage level higher than 10t/y.this test is waived if the substance is unlikely to cause aquatic toxicity, for example, if it is insoluble in water; we assume that this is the case for 10% of chemicals.eCB assumes that data exist for 49% of chemicals.the test is not necessary when a long-term toxicity study is available; eCB expects this for 7% of chemicals, but this should be typically on substances where the short-term test is also available.this means that the test will be carried out for 45.9% of chemicals.According to the of nervous system involvement in adult studies e.g.repeated dose toxicity studies 3) Have a mode of action that has been closely linked to neurotoxic or developmental neurotoxicity effects evaluations of repeated-dose toxicity studies (Bitsch et al., 2006) suggest about 10% of chemicals cause neurotoxic effects, thus we assume that in half of the cases (5%) DNt studies will be considered and carried out in 2.5%, as suggested in the OeCD tG 426.this is disputed by some practitioners, but optimistically we will assume that this is possible in half of the cases, resulting in a 50% cost reduction and no additional animals.Furthermore, it is suggested that "evidence of endocrine disruption seen in a repeated dose toxicity study provides a trigger for the conduct of a more comprehensive study, for example a two-generation study."evidence of endocrine disruption and pathology to endocrine glands is again seen for about 10% of chemicals (Bitsch et al., 2006), which might result in similar assumptions in 2.5% of two-generation studies triggered by repeat-dose studies in Annex VIII and Ix.
the guidance does not identify any in vitro ("no officially adopted eU or OeCD test guideline") or (Q)SAR ("cannot be adequately covered by a battery of (Q)SAR models") substitutes for testing.Reproductive toxicity is especially difficult for structure-based predictions, since sometime even enantiomers or slightest chemical modifications change the toxic profile, as is well known for hazardous substances such as thalidomide or vitamin A derivatives.In fact, a systematic assessment of the most promising commercial (Q)SAR programs led to disappointing results (Maslankiewicz et al., 2005) the software program DeReKfW has been challenged with the around 100 reproductive toxicants included in Annex I of Directive 67/548/ EEC.About 90% of chemicals classified for "impaired fertility" and 81% of chemicals that cause harm to the unborn child were not detected.the tSCA (toxic Substances Control Act) chemical category list of the US ePA's new chemical program failed in 77% of cases to detect EU classified chemicals causing adverse effects to mammalian fertility, and 82% of developmental toxicants have not been correctly identified.
In vitro approaches are furthered by the eU ReProtect project (Hareng et al., 2005), but a replacement strategy is not foreseeable.Currently, the most important approach is the ongoing validation of replacing the two-generation test by an extended  et al., 2004).We further assume that in half of the cases the validated threshold-step-down approach will be used, which reduces animal use by 60%.
For substances covered by Annex Ix, long-term toxicity study is always required and no additional test is added in Annex x.For long-term toxicity in fish, OECD TG 210, 212, and 215 are the preferred methods, as they consider different sensitive life stages of fish.The most used method is OECD TG 210, with an average cost of 26,300 euro and 400 fish per tested chemical.states, resulting in about 68,000 substances.It also used minimal animal numbers per test and neglected most triggering of additional tests by suspected CMR (carcinogen/mutagen/reproductive toxicant) and biopersistent chemicals as well as triggers from other in vitro/in vivo/in silico findings or yet not defined testing demands (endocrine disruption, respiratory irritation, respiratory sensitization, developmental neurotoxicity, etc.).A number of assumptions had to be made, and were reviewed by the practitioners named in the acknowledgement.the overall result suggests a demand of 54 million vertebrate animals and testing costs of 9.5 billion euro.Because of the nature of assumptions, this represents a best-case scenario.this number has to be compared to about 90,000 animals used for testing of new currently available.Due to the small numbers involved, the high uncertainty connected to the present assumption does not affect the final results.

Summary of assumptions and results
tabs. 17 and 18 summarize all the assumptions of the previous paragraphs.It is quite clear that the most demanding studies are in the area of reproductive toxicity testing, with about 90% of all animal use and 70% of the required costs for registration.this study used rather conservative estimates, i.e. adjusting the number of chemicals falling under ReACH from 1994 due to the growth of chemical industry and the eU member man health and the environment from unwanted consequences of exposure to chemicals.the challenge will be to do it sensibly within the context of ReACH while using all the information and experience we have and recognizing that most chemicals have been produced and used safely for many years without extensive testing on animals.chemicals per year in europe.ReACH aims to complete data collection by 2018.At that point, Annex VII and VIII chemicals, which do not require permission to carry out suggested animal tests, have to be registered.In parallel, for Annex Ix chemicals registered by 2013, permission to carry out suggested animal tests shall be given within two years, and execution within the following years would be expected.thus, most animal testing will start around 2012, i.e. two years after the registration of Annex x substances, and the majority of these tests should be completed in the relatively short period of 6 years.theoretically, this means about 100-fold increase in animal use for chemical testing and about doubling of overall animal use in europe.this clearly challenges the feasibility of the program.Without a major investment into high-throughput methodologies this program cannot be completed in a reasonable time frame.It is obvious that such methods will not be available by 2012.
Notably, in the areas of highest contribution to animal use and costs, i.e. reproductive toxicity, repeat dose toxicity, and longterm toxicity in fish, no relevant alternative approaches have emerged.At least no method is under validation, which means that no validation / peer-review / acceptance can be expected before 2018.the notable exception is the opportunity to change from the two-generation study to an extended one-generation study (Bremer et al., 2007, Spielmann and Vogel, 2007, Moore et al., 2009).this would reduce animal use for this test by 40-60%.this means about a 15% reduction in total animal numbers.this will only impact the cost of tests if the extensions of one-generation study, i.e.DNt and immunotoxicity, are not made mandatory for chemicals.Otherwise the costs of the twogeneration-study and the extended-one-generation-study will not differ substantially.

Conclusions
the re-evaluation of animal numbers and costs of ReACH demonstrates that estimates must be increased substantially.Preregistrations for ReACH clearly exceeded expectations and several contributing factors, such as expansion of eU member states and growth of the chemical industry, were neglected in the earlier assessments.the guidance for industry published by eCHA does not offer the expected waiver opportunities and the availability of safety assessments from other sources was probably too optimistic.At the same time, despite an obvious stimulus, the development and validation of alternative methods has been minimal for the most complex endpoints.Successes in areas like topical toxicities, acute toxicity, acute fish toxicity and sensitization do not translate to major savings because of the dominance of reproductive toxicity testing requirements.this lack of progress is especially true in the most demanding areas of reproductive toxicity and long-term fish toxicity, with the notable exception of the extended one-generation test to replace the two-generation test.the feasibility of ReACH on this basis has to be questioned.It seems to be inevitable to either prioritize testing requirements or reconsider testing approaches as currently suggested by ePA in the US (ePA, 2009b).It is beyond dispute that the primary aim of ReACH is protecting hu-

3
Number of in vivo toxicity teststhe types of in vivo toxicology tests required for filling the CSR of a substance are described in different ReACH Annexes according to tab. 8, i.e. total production or marketing volume in europe determines the testing requirements which are then modified by specific toxicity and use profiles of the substances.ReACH immediately asks for the results of tests described in Annexes VII and VIII, while the tests under the provisions of Annexes Ix and x are proposed by the submitter and performed only after receiving the authorization by eCHA.Annex VII requires only skin sensitization and acute toxicity as in vivo tests and no CSR.Acute toxicity study by oral route is implemented in Annex VIII by another route, either dermal or inhalation.Moreover, the short term repeated dose toxicity study can trigger further studies if there are indications of toxicity of particular concern.the screening developmental toxicity the strategy for reducing the number of tests performed for each substance is well accepted by ReACH.Annex xI outlines Tab.9: Existing data for each endpoint as foreseen by Pedersen et al. (2003), based on data from RPA & Statistics (2001) Noteworthy, long-term bird studies were not included in the previous reports.
Annex I of the eU Dangerous Substance Directive 67/548/ eeC • Proposition 65 list of the State of California Office of Envi- ronmental Health Hazard Assessment • european Chemical Substances Information System and IUClID Chemical Data Sheet Information System • US National toxicology Program (NtP) database of repro- ductive and developmental toxicity study abstracts • US ePA's Integrated Risk Information System database and peer-reviewed open literature

Comparison of number of preregistered substances and estimation done by Pedersen et al. (2003)
Data from ECHA Press Release (2008) and ECHA Press Release (2009) as well as Pederson et al. 2003.

Tab. 4: List of preregistered substances according to their EC classification
Data from ECHA Press Release (2008) and ECHA Press Release (2009).

Total number of chemicals requiring registration according to REACH estimated by RPA & Statistics, 2002 Foreseen Total number of registrations
4,381 substances.elINCS substances have been already notified under Directive 67/548/EEC and the dossier can be accepted as registration for ReACH without requiring further in vivo testing.

Registration deadline With CAS number Without CAS number Total without regular EC number
Pépins de raisin: It is the French name for grapeseed oil, which is the oil extracted from grapeseeds.there are many other entries about the processing of grape, some of them differing only in the spelling, such as Pulpe Understanding the meaning of this entry is impossible.Presumably it deals with a recovering process.this section of ReACH is still vague and a new guideline about waste and recovered substances is expected shortly.there are about 20 entries with very similar substance names, differing only in typos of the substance name, indicating that they all refer to the same product.d. eC 917-706-8 Eisen: Totally inadequate substance definition.

Tab. 7: Foreseen number of expected full registration dossiers
Different scenarios by marketed quantities (i.e.basic testing demands), with (1) considering all preregistrations; (2) excluding likely mistakes as explained in the text; (4) estimates by Pederson et al. (2003) and (3) correcting scenario 4 with 97% growth of chemical industry in 14 years and 18% by expansion of the EU.

: In vivo tests required in Annexes VIII through X of the REACH legislation
Pedersen et al., 2003)have considered both the availability of data as recorded in the HPV (High Production Volume) database and the data collected by RPA and Statistics through a survey conducted on industries and other organizations.RPA and Statistics (also found in tab. 2 inPedersen et al., 2003)grouped tests in Base set, level 1, and level 2 data, as described in Directive 67/548.This classification now approximately corresponds in ReACH to Annex VII and VIII, Annex Ix and Annex x, respectively.

Expected impact from alternative approaches: emerging validated/not accepted and methods under validation
In 2004, van der Jagt et al. published a report about how alternative approaches can reduce the use of test animals under REACH.The first step is focused on (Q)SAR (Quantitative Structure Activity Relationship

Tab. 14: Assumptions for the requests of new in vivo Acute Toxicity studies
Cost and minimum animal number by OECD test guideline as well as estimated percentage of substances per tonnage level to be subjected to the different acute toxicity tests.See text chapter 4.3.

Tab. 15: Assumptions for the requests of new in vivo Repeated Dose Toxicity Studies Cost
calculate 3,200 and minimum animal number by OECD test guideline as well as estimated percentage of substances per tonnage level to be subjected to the different repeated dose toxicity tests.See text chapter 4.4.

Tab. 16: Assumptions for the requests of new in vivo Reproductive Toxicity Study Cost
and minimum animal number by OECD test guideline as well as estimated percentage of substances per tonnage level to be subjected to the different acute toxicity tests.See text chapter 4.7.