First Evaluation of the Use of “Non-animal” Methods to Meet the Requirements of the EU Chemicals Directive REACH

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Programs & Policies - EU

First Evaluation of the Use of “Non-animal” Methods to Meet the Requirements of the EU Chemicals Directive REACH

Horst Spielmann, Freie Universität Berlin

Published: August 17, 2011

About the Author(s)
Prof. Dr. Horst Spielmann
Zerbster Str. 22
12209 Berlin, Germany
phone: +49-30-7118661
fax: +49-30-71581672
mobile: +49-1773144554
e-mail: horst.spielmann@fu-berlin.de
ECHA, the European Chemicals Agency in Helsinki (Finland), has on June 30, 2011, published two reports: one on the operation of the new EU chemicals regulation REACH (Registration, Evaluation and Accreditation of Chemicals), and one on the use of alternatives to testing chemicals on vertebrate animals. These reports are legal requirements of the REACH Regulation. ECHA’s report on REACH presents a statistical analysis of the data submitted in 24,560 registration dossiers between 1 June 2008 and 30 November 2010, covering non-phase-in (new) and phase-in (existing) substances and focusing on those manufactured or imported at above 100 tonnes per year (tpa), because these have the highest data requirements. The number of substances covered by the deadline was 4,300, of which 3,400 were phase-in (existing) substances.

In order to be able to correctly interpret the outcome of the ECHA report on the use of alternatives, some specific results of the statistical report on the operation of ECHA has to be taken into account. Therefore, a short summary of the statistical report will be given first and then the report on the use of alternatives will be given.

1. ECHA Report on “The Operation of the REACH Regulation”

REACH is designed to gather data to be submitted in dossiers when companies register under each of the three deadlines. At the same time, another strong principle of the Regulation is that new animal testing should be carried out only as a last resort. One of the ground breaking elements of REACH is that it explicitly gives companies flexibility to employ non-animal-test methods and other approaches to fulfil their information requirements and to directly support safety claims about their substances.

The overarching message of the report is that the REACH Regulation is working well and that the various actors responsible for the work are responding as required. This is largely attributable to the commitment and collaborative work between industry, stakeholders, the Member States, the European Commission and ECHA.

At the first registration deadline of 30 November 2010 industry had registered 4,300 substances in 24,560 dossiers, 3,400 of them were phase-in (existing) substances. These numbers are much lower than anticipated, in particular when taking into account that 140,779 phase-in substances had been registered prior to November 30, 2008.

1.1 Data Sharing: One Substance, One Registration

Data sharing is one of the core principles in the REACH Regulation. By submitting dossiers jointly and sharing information on substances, companies increase the efficiency of the registration system, reduce costs and avoid unnecessary testing on vertebrate animals. As a consequence, for each substance only a single dossier should be submitted. To achieve this goal for each substance, companies should establish a substance information exchange forum (SIEF) and the dossier should be submitted by the Lead Registrant. The number of companies forming a SIEF varied considerably, e.g. for 146 substances SIEFs of more than 1000 companies have been established. This approach to data sharing has reduced the cost of registering, but confidentiality and legal issues were unprecedented and quite challenging. In the report, ECHA concludes that the REACH concept of data sharing worked well and the first round of submissions was, therefore, a success.

However, the complexity of substance identification for phase-in (existing) has been underestimated in REACH. Dossier evaluation has indicated that substance identity in registration dossiers is often not adequately described for phase-in (existing) substances. ECHA and the Commission should investigate how the data-sharing procedures can be made more transparent and how to promote best practice for data-sharing before the forthcoming registration deadlines.

1.2 Evaluation of Registration Dossiers

The competence of ECHA in evaluating dossiers is rather limited since “The REACH Regulation requires that ECHA carries out COMPLIANCE CHECKS on at least 5% of the total number of registration dossiers received for each tonnage band.”

Taking into account that 3,400 phase-in (existing) substances have been registered and that ECHA has performed compliance checks on 5% of them, so far only 170 chemical have been evaluated. From the remaining of 900 phase-out (new) substance, only 45 have been evaluated.

As a result of the first compliance checks, ECHA reports that “these initial compliance checks have indicated that a significant proportion of dossiers have shortcomings and still need to be improved with further information” and that “the scope of the compliance check process limits ECHA to requesting missing information.”

2. The ECHA Report “On the Use of Non-animal Test Methods”

The report is the first provided by ECHA on the use of alternatives to testing on animals since REACH came into effect. The registration dossiers for high tonnage substances submitted from 1 June 2008 until 28 February 2011 were analysed to get a picture of the use of animal studies and non-animal methods used.

Registrants made full use of the options available in REACH to use alternatives to testing on vertebrate animals. This includes, for example, using existing studies or applying non-test methods to predict properties of substances instead of experimental testing. The report shows that so far very few new animal studies were conducted for the purpose of registering phase-in (existing) substances.

To keep this review focused on the use of alternatives to the use of laboratory animals, only endpoints related to human health effect are covered. Studies conducted to meet the requirements for environmental toxicty were excluded, e.g. bioaccumulation, acute and long-term toxicity to fish.

2.1 Data Used in the ECHA Analysis

From the original 24,560 registration dossiers, 17,062 dossiers were identified to be registration dossiers with a tonnage band at or above 100 tpa. It was necessary to exclude dossiers for ‘chemical categories’ (i.e. IUCLID category dossiers) (n=568 or 2.3%) from the in-depth analysis due to the complex endpoint interrelationship between dossiers that currently did not allow a reliable data analysis to be performed. This left 16,494 dossiers in the data set for analysis. Of these dossiers, only the lead registrant’s dossiers and dossiers for individual registrations contained endpoint information for the registered substances, resulting in 1,862 phase-in (existing) substances The ECHA analysis only takes into account high volume chemicals produced at ≥1,000 tpa for a total number of 1,504 substances.

2.2 Approaches Used to Analyse the Use of Non-animal Alternative Methods

Since ECHA has only performed COMPLIANCE CHECKS and not performed an in depth analysis, an entry listed as experimental study under an endpoint does not mean that the information requirement is filled according to the requirements of REACH.

ECHA has used two approaches to analyse the use of alternatives, the Endpoint Study Record (ESR) approach and the substance approach.

The ESR approach consists of the analysis of all endpoint study records (experimental and several non-experimental methods) submitted for the 1,862 dossiers for a given endpoint. For each endpoint, more than one or even many endpoint study records are possible and were summarised for these dossiers (see below). The ESR approach provides the overall quantitative picture of options used by registrants for dossiers within the scope of this analysis.

It is of further interest to analyse at the substance level to determine how the registrants used alternative approaches (the substance approach). Such an analysis provides the relative proportions of the principal options used by registrants to fill the information requirements per endpoint. These options have been categorised as testing proposals, experimental studies and alternative methods.

2. 3 Results Obtained with the ESR Approach
2.3.1 Terminology Used in the ESR Approach

For the purpose of the ESR approach, the following groups of options have been used in the ECHA report:

  • Alternatives to testing on animals: ECHA uses the term “alternatives”for all “non-experimental” studies while the term “experimental studies” includes both in vivo and in vitro.
  • Experimental studies (ES): classified by the registrant as “experimental result” from the pick-list of options in the field “study result type.” The term ES is used for both in vitro and in vivo studies.
  • Testing proposal (TP): classified by the registrant as “experimental study planned.”
  • Read-across (RA): classified by the registrant as read-across.
  • IUCLID flags to omit the study (FO) (= waiving): selected by the registrant to omit the submission of the required data by choosing “data waiving.”
  • Weight of Evidence (WE): classified by the registrant as weight of evidence.
  • (Q)SAR studies (QS): classified by the registrant as “(Q)SAR studies.”
  • Miscellaneous (MS): classified by the registrant as “other.”
2.3.2 Acute Toxicty: All Routes

There were 1,504 dossiers covering phase-in (existing) substances and 12,874 (100%)data sets (ESR) submitted for this endpoint. The ESR distribution showed the following pattern:ES (in vivo) 56.9%, RA 21.4%, FO (waiving) 9.2%, WE 8.7%.

The majority of acute toxicity ESRs were data from in vivo experimental studies and neither data from in vitro experimental studies nor (Q)SAR data were submitted; 43.1% of the ESRs were non-experimental alternatives.

2.3.3 Skin Irritation/Corrosion
2.3.3.1 Skin Irritation In Vitro

For this endpoint, validated in vitro methods are available (OECD TG 439). For the 1,504 phase-in (existing) substances, 329 ESR (100%) were submitted. The ESR distribution showed the following pattern: ES (in vitro) 76.6%, RA 11.9%, FO (waiving) 0.6%, WE 10.6%.

The majority of in vitro skin irritation data were obtained in studies performed most recently, since this method was accepted by the OECD in 2010. The high percentage of in vitro experimental studies of 76.6% is remarkable. The percentage of non-experimental alternatives was less than 25% and no (Q)SAR data were submitted.

2.3.3.2 Skin Irritation In Vivo

The registrants submitted 5,216 ESRs (100%) for in vivo skin irritation for the 1,504 phase-in (existing) substances. The ESR distribution provided the following result: ES (in vivo) 64.1%, RA 21.%, FO (waiving) 4.1%, WE 7.7%.

The majority of in vivo skin irritation data were obtained in studies performed before an in vitro skin irritation test had been validated and accepted. No (Q)SAR data were submitted and 35.9% were non-experimental alternatives.

2.3.4 Eye Irritation
2.3.4.1 Eye Irritation In Vitro

For this endpoint, a positive outcome from in vitro assays such as the bovine corneal opacity and permeability (BCOP, OECD TG 437) or isolated chicken eye (ICE, OECD TG 438) tests is sufficient to classify severe eye irritants. For the 1,504 phase-in (existing) substances, 172 ESR (100%) were submitted. The ESR distribution showed the following pattern: ES (in vitro) 86.6%, RA 7.0%, FO (waiving) 0.6%, WE 2.9%.

The majority of in vitro eye irritation data were obtained in studies performed most recently since the in vitro eye irritation tests were accepted by the OECD in 2009. The high percentage of experimental studies 86.6% is remarkable. Less than 15% non-experimental alternatives were submitted and no (Q)SAR data.

2.3.4.2 Eye Irritation In Vivo

The registrants submitted 4,221 ESRs (100%) for in vivo eye irritation. The ESR distribution provided the following result:ES (in vivo) 64.3%, RA 20.9%, FO (waiving) 5.2%, WE 6.6%.
The majority of in vivo eye irritation data were obtained in studies performed before an in vitro eye irritation test had been accepted by the OECD. No (Q)SAR data were submitted and 35.7% were non-experimental alternatives.

2.3.5 Skin Sensitisation

All of the standard skin sensitisation test methods, for which EU or OECD TGs are available, are in vivo tests. They include the guinea pig maximisation test (GPMT), the occluded patch test of Buehler and the murine local lymph node assay (LLNA, OECD TG 442A & 442B).

Today the LLNA is the first choice method for in vivo testing. However, submitted historical data for this endpoint comprised LLNA, GPMT and Buehler test data.

For the 1,504 phase-in (existing) substances, 3,754 ESRs (100%) have been submitted. The ESR distribution provided the following result: ES (in vivo) 55.4%, RA 20.8%, FO (waiving) 7.0%, WE 13.7%.

The majority of ESRs were data from in vivo experimental studies (55%) and no (Q)SAR data were submitted for this endpoint while 44.6% were data from non-experimental alternatives. Quite unexpectedly there were 21 entries for in vitro skin sensitisation studies. Further analysis revealed that in most cases, these entries were (in vivo) LLNA tests that were misclassified by registrants as in vitro tests.

2.3.6 Repeated Dose Toxicity

In vitro methods have not been validated for repeated dose toxicity; 10,700 ESRs (100%) have been submitted for the 1,504 phase-in (existing) substances. The ESR distribution provided the following result: ES (in vivo) 42.1%, RA 28.1%, FO (waiving) 18.8%, WE 6.6%, MS 4.4%.

It is quite surprising that less than 50% of ESRs were from in vivo experimental studies and no (Q)SAR data were submitted. As a consequence more than 50% of ESRs were non-experimental alternatives.

2.3.7 Genetic Toxicity

According to the REACH Regulation only in vitro mutagenicity tests are needed for the core data. Some in vivo confirmatory mutagenicity studies may be necessary as higher-tier studies to be conducted after the testing proposals have been approved.

2.3.7.1 Genetic Toxicity In Vitro

For genetic toxicity in vitro, 10,322 ESRs (100%) were submitted for the 1,504 phase-in (existing) substances. The ESR distribution showed the following pattern: ES (in vitro) 57.2%, RA 22.0%, FO (waiving) 3.8%, WE 12.1%, OS 0%, MS 4.8%.

The majority of ESRs were data from in vitro experimental studies (57.2%) and no (Q)SAR data were submitted, while 42.8% of the ESRs were non-experimental alternatives.

2.3.7.2 Genetic Toxicity In Vivo

The registrants submitted 3,533 ESRs (100 %) for genetic toxicity in vivo. The ESR distribution had the following result: ES (in vivo) 52.4%, RA 24.8%, FO (waiving) 6.3%, WE 11.0%, QS 0%, MS 5.0%.

Although only in vitro genotoxicity data are required according to the REACH Regulation, registrants have submitted existing in vivo genotoxicity data from their files; 47.6% of ESRs were from non-experimental alternatives.

2.3.8 Toxicity to Reproduction
2.3.8.1 Toxicity to Reproduction (Screening Tests and 1- & 2-generation Studies)

Reproductive toxicity addresses two related endpoints, which are usually tested separately: a prenatal developmental toxicity study and a reproduction toxicity study covering one or more generations. Alternative in vitro tests are currently not able to adequately predict the complex endpoints covered by the two in vivo tests.

For the substances produced at 10-100 tpa, a reproduction/developmental toxicity screening test (ref. OECD TG 421 or 422) is usually required to meet the core data requirements. For substances manufactured or imported between 100-1000 tpa, in addition a prenatal developmental toxicity study (ref. OECD 414) is required, and for substances produced at higher annual tonnage a two-generation reproduction toxicity study (ref. OECD TG 416, EU B.35) is required.

The registrants submitted 3,535 ESRs (100 %) for reproductive toxicity of the 1,504 phase-in (existing) substances. The ESR distribution provided the following result: ES (in vivo) 31.7%, TP 4.2 (not yet decided), RA 22.8%, FO (waiving) 25.6%, WE 12.1%, QS 0.1.%, MS 2.5%.

Less than 1/3 (31.7%) of ESRs for this endpoint were from in vivo experimental studies and no (Q)SAR data were submitted. Therefore, more than 2/3 (68%) of ESRs were non-experimental alternatives. ECHA has not yet evaluated whether the read-across and grouping approaches and justifications for omitting the information are in line with the REACH requirements.

2.3.8.2 Developmental toxicity

The registrants submitted 4,217 ESRs (100 %) for the endpoint developmental toxicity. The ESR distribution showed the following result: ES (in vivo) 42.3%, TP 3.6 (not yet decided), RA 29.7%, FO (waiving) 10.9%, WE 10.7%, QS 0.2%, MS 2.6%.

The percentage of in vivo experimental studies was less than 50% for this endpoint and no (Q)SAR data were submitted. More than 50% of the ESR were non-experimental alternatives. ECHA has not yet evaluated whether the read-across and grouping approaches and justifications for omitting the information are in line with the requirements of REACH.

2.3.9 Carcinogenicity

There are no accepted in vitro tests for this complex endpoint. The 2-year cancer assay in rodents is typically conducted to evaluate the cancer hazard and potency of a substance.

The registrants submitted 3,559 ESRs (100 %), and the ESR distribution provided the following result: ES (in vivo) 38.7%, TP 0.1 (not yet decided), RA 27.9%, FO (waiving) 14.9%, WE 12.2%, QS 02.%, MS 6.1%.

Only a little more than 1/3 (38.7%) of ESRs for this endpoint were from in vivo experimental studies, as a consequence almost 2/3 (61.3%) of ESRs were non-experimental alternatives.

2.3.10 ECHA’s comment on the use of alternatives based on ESR analysis

ECHA concludes that, in the context of the overall number of all ESRs extracted from registration dossiers of all tonnage bands, and both phase-in (existing) and non-phase-in (new) substances, new studies represented less than 1% of the total ESRs extracted for these endpoints. Therefore, it can be concluded that registrants mainly used old experimental data as well as the options for the adaptation of the standard information requirements and other alternatives before electing to conduct new experimental in vivo or in vitro studies to meet their obligations and make their registration dossiers compliant under REACH.

2.4 The “Substance Approach” to Analyse the Use of Alternative Methods

For most endpoints several ESRs have been submitted, therefore, this approach does not provide information on the actual numbers of experimental and non-experimental studies submitted for individual substances. An entry as experimental study under an endpoint does not mean that the information requirement is met according to REACH requirements. This is specifically relevant for repeated dose toxicity, toxicity to reproduction and developmental toxicity.

Acute toxicity:

In 85% of cases, this endpoint was filled with in vivo experimental data. The rest was filled with information using only non-experimental alternative options.

Skin irritation:

In 78% of the cases the skin irritation endpoint was filled with combined in vitro and in vivo experimental data (2.3.3.1 & 2.3.3.2), while in 22% only non-experimental alternative options were used.

Eye irritation:

The eye irritation endpoint was covered in 75% of cases by in vitro or in vivo experimental data and in 25% non-experimental alternative options were used.

Skin sensitisation:

For this endpoint 63% of data submitted referred to in vivo experimental studies and 37% by non-experimental alternative options.

Repeated dose toxicity:

Around 67% of the submitted data were in vivo experimental studies and 26% were non-experimental alternative options. In 7% of cases, proposals for testing were submitted.

Genetic toxicity:

The genetic toxicity in vitro endpoint was covered by experimental data in more than 77% of cases, while 32% used non-experimental alternative options. Genetic toxicity in vivo data were available for 41% of the cases. To cover this endpoint today there is a shift toward using in vitro experimental data.

Toxicity to reproduction and pre-natal developmental toxicity:

Almost 42% of the analysed phase-in (existing) substances had experimental in vivo data on toxicity to reproduction – both from screening tests and two-generation studies, while 48% of the cases used non-experimental alternative options. In 10% of cases, proposals for additional in vivo testing were submitted.

For prenatal developmental toxicity, experimental in vivo studies were available for 47% of substances – both from screening tests and developmental toxicity tests. In 43%, non-experimental alternative options were used. In 10%, proposals for additional in vivo testing were submitted.

Carcinogenicity:

For carcinogenicity, the ECHA report on the use of alternatives does not provide an analysis according to the “substance approach.”

2.5 ECHA’s Comments on Data Submitted to Meet REACH Requirements

The number of studies using animals conducted or proposed for the purpose of REACH are lower than expected in previous publications predicting animal tests to be performed for REACH Regulation. The reasons are that the number of high tonnage level substances is lower than expected, data sharing was working well between the registrants, and the adaptation possibilities have been fully used by the registrants. However, in the context of the overall number of all ESRs extracted from registration dossiers of all tonnage bands, and both phase-in (existing) and non-phase-in (new) substances, new experimental studies – both in vitro and in vivo – represented less than 1% of the total ESRs extracted for these endpoints. Therefore, it can be concluded that registrants mainly used old experimental data as well as the options for the adaptation of the standard information requirements and other alternatives before electing to conduct new studies to make their registration dossiers compliant under REACH.

REACH Annexes require the approval of testing proposals by ECHA before being conducted. In total 107 studies appeared to be conducted in the absence of approval by ECHA. ECHA also noted that for some higher tier test requirements, screening studies had been submitted in place of the actual test(s) specified in REACH. This may result in additional, new animal testing compared with the results provided in the first ECHA report.

2.6 Discussion

When the new EU REACH legislation for chemicals was discussed by the EU Parliament, animal welfare aspects had a very high priority among legislators, the general public and the EU Commission.

When assessing this ECHA report one has to take into account that “The REACH Regulation requires that ECHA carries out COMPLIANCE CHECKS on at least 5% of the total number of registration dossiers received for each tonnage band.” Thus the analysis provided is not based on an in-depth scientific analysis of the REACH registrations.

It is also important to recognize that the dossiers are owned by the registrants, usually industry, and that the submitters are, therefore, responsible for the quality of the data and the safety measures (CLP = classification, labelling and packaging) that have to be implemented. Since ECHA has only the competence to check the compliance of the data submitted, there is still a high level of uncertainty about the quality of the submitted experimental and non-experimental data.

In the two reports, ECHA is using the term “alternatives” for all “non-experimental” studies. Thus, in the analysis, the term “experimental studies” is used for both “in vivo” and “in vitro” studies and ECHA is using the term “alternatives” basically for “all non-experimental” approaches to meet the REACH requirements. This includes “read-across” and “grouping,” “waiving,” “weight of evidence,” (Q)SAR as well as “proposals for additional testing.”

Both from the scientific and animal welfare perspective, the most important results published in the ECHA report on alternatives for the high production chemicals are:

  1. Industry has successfully met the unprecedented challenge of data sharing, which has significantly contributed to avoid unnecessary testing in animals.
  2. New experimental in vivo or in vitro studies that were performed to meet the data requirements for REACH represented less than 1% of the total experimental study records (ESRs).
  3. Almost all data requirements could be met by using existing data from the files of registrants.
  4. The majority of data submitted are existing (old) data from existing experimental studies in animals from the files of industry.
  5. The classical experimental “in vitro” toxicity tests played a minor role and were only used for a limited number of endpoints where validated OECD methods were available, e.g. for skin and eye irritation.
  6. For predicting substance toxicity “read-across” was the second most used approach followed by “weight-of-evidence” and “waiving.”
  7. (Q)SAR was almost not used at all, even for endpoints which seem promising, e.g. genotoxicity and skin and eye irritation/corrosion

When the REACH policy was discussed in the EU Parliament, several expert groups had predicted that the new EU chemicals legislation would lead to an unprecedented increase in the number of experimental animals used for toxicity testing, in particular for long term toxicity, e.g. systemic repeated dose toxicity, reproductive and developmental toxicity and carcinogenicity. Even quite conservative estimates from the German Federal Institute for Risk Assessment BfR (Höfer et al., 2004) and from the Joint Research Centre of the EU (van der Jagt et al., 2004) estimated animal numbers between 4 and 45 Mio. These and many other authors did not take into account the wealth of information from the files of industry and the extensive use of the non-experimental estimates based on “read-across,” “weight-of-evidence” and “waiving.”

In essence it has been a goal of the REACH legislation to make the use of chemicals in Europe and beyond safer, in particular by evaluating toxicity data of existing chemicals, which were in the files of industry. In essence REACH is about providing toxicity data of chemicals to regulators within a very short time frame to register, evaluate and accredit the most hazardous ones. Since industry has quite substantially cooperated and provided existing data to ECHA, the safety of chemicals can be effectively assessed and safety measures can be implemented while hardly any additional in vivo studies in animals had to be conducted. This is a major achievement to which scientists in industry, academia and regulatory agencies have contributed.

Outlook: REACH as a Challenge for Safer Chemicals in the 21st Century

On the weekend before the final formal approval of the new REACH policy by the EU Parliament in 2003, the heads of the three major member states of the EU, Blair (UK), Chirac (France) and Schroeder (Germany) met in Berlin and came up with a very strong statement against the proposed REACH legislation, stating that it would jeopardise Europe’s position in a globalised economy, and that the main competitors outside Europe would not be restricted by legal requirements and be able to sell their chemicals cheaper. Experience has shown that in a globalised world the REACH initiative was not a hurdle for Europeans, but instead citizens and legislators outside Europe are following the European example. Thus far, REACH has been an outstanding start for safer chemicals in the 21st century.

©2011 Horst Spielmann

References
ECHA. (2011). The operation of REACH and CLP. Available here: http://echa.europa.eu/doc/117reports/operation_reach_clp_2011_en.pdf.

ECHA. (2011). The use of alternatives to testing on animals for the REACH Regulation 2001. Available here: http://echa.europa.eu/doc/117reports/alternatives_test_animals_2011_en.pdf.

Höfer, T., et al. (2004). Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation. Archives of Toxicology. 78: 549-564.

van der Jagt, K., et al. (2004). Alternative approaches can reduce the use of test animals under REACH. JRC Report EUR 21405 EN. Available here: http://ecb.jrc.it/documents/REACH/publications.