The aim of environmental enrichment is to alter the environment of a captive animal in a way that results in improved mental and physical welfare. The technique has been utilised effectively for many years for captive mammals in a variety of settings. However, until now it has never been considered as a way of improving the welfare of aquatic animals such as fish. Fish that are used in regulatory toxicology studies are at present maintained solely in barren tank environments. Little is known about how these types of environments affect the well-being of the animals residing there and whether they impact either physiological heath or behavioural repertoire. This thesis aims to address this gap in the knowledge regarding the potential for environmental enrichment to improve the welfare of fish used in regulatory toxicology. More specifically it looks at two types of enrichment and the effects of these on the commonly used model species, the zebrafish (Danio rerio). The first type of enrichment studied was glass rod structures of varying heights provided to increase tank complexity and provide refuge. The glass structures did not produce any quantifiable benefits in unstressed fish and appeared to delay the formation of stable social hierarchies. When fish were stressed by a period of chasing, the presence of the glass rods appeared to reduce the magnitude of the cortisol response. Whilst this could be viewed as a potential benefit, it was felt that it would not outweigh the costs of this type of enrichment. The second type of enrichment studied was provision of airstones. Again, no clear evidence was found that fish in tanks with airstones experienced an improvement in welfare. The main observation was the vast increase in mortality in tanks containing these airstones, in particular, those of a smaller size. Regardless of the physiological cause underlying this result, this can only be viewed as a negative consequence and one that appears to rule out airstones as an effective form of enrichment for this species and strain of fish. It was also observed that both stress and the presence of enrichment influenced the absolute deviation from the mean in several endpoints. Since changes in endpoint variation will have effects both on the number of animals required to statistically measure environmentally relevant effects this is a factor that should be considered when researching methods of environmental enrichment. Finally, results from these studies suggest the possibility that laboratory zebrafish do not require the addition of environmental enrichment to tanks in order to promote maximum welfare. Furthermore, as considerable costs would be involved in implementing many types of enrichment (relating to manufacture, cleaning, incompatibility of results with previous studies etc.) it is likely that observed benefits would have to be both substantial and well established in order for changes in regulatory guidelines to take place. For a species such as zebrafish that are extremely easy to breed and maintain in the laboratory with minimal amounts of disease, social problems or mortalities, it may be that current conditions are satisfactory.

<p>For the majority of the estimated 70,000 industrial chemical substances available on the European market today there is not enough information to enable a reasonably complete assessment of the risks that they might pose to man and the environment. Any strategy for the generation of additional data for these substances should aim at making testing as efficient as possible taking into account environmental and health protection, time, monetary cost and animal welfare. To achieve this, appropriate priority setting rules are needed.</p><p>The main criterion currently used for regulatory priority setting for testing of industrial chemicals is production volume; the higher the production volume, the more information is required. This was also the main criterion in the former legislation, preceding REACH (Registration, Evaluation and Authorisation of Chemicals). The aim of this thesis is to evaluate other priority setting criteria and their implications for risk management, in particular classification and labelling.</p><p>The first paper in this thesis includes a study of the<i> efficiency ratio</i> for some of the tests required for the notification of new substances, i.e. the ratio between the likelihood that the test will lead to a classification, and the monetary cost of performing the test. The efficiency ratio was determined for the standard tests for acute oral toxicity, irritation, sensitisation and subacute toxicity using data from 1409 new chemicals notified in Europe between 1994 and 2004. The results of this investigation suggest that, given limited resources for testing, it is more efficient to perform acute toxicity tests on a larger number of substances rather than to perform additional subacute toxicity studies on the substances already tested for acute toxicity.</p><p>The second paper included in this thesis, reports the results from a comparative study of the bioaccumulating properties of substances being (a) classified as carcinogenic, mutagenic and/or toxic to reproduction (CMR-substances), or (b) classified as acutely toxic or (c) unclassified. The purpose of this investigation was to evaluate potential consequences of prioritising bioaccumulating chemicals for evaluation and testing, as this is one of the strategies prescribed in REACH. The results of this study suggest that bioaccumulating substances are neither over- nor underrepresented among the CMR-substances. This result lends support to the use of the bioconcentration factor for priority setting.</p><p>The studies reported in this thesis utilize existing data on classification of substances as an indicator of the outcome of the risk assessment process, relating priority setting methods to the risk management measures that they give rise to. To the best of my knowledge there are still only very few studies published that address the issue of priority setting in chemicals control using this approach, and in my view there is need for more studies of priority setting methods and a further development of priority setting strategies that are science-based.</p>

<p>Occupational Exposure Limits (OELs) are used as an important regulatory instrument to protect workers’ health from adverse effects of chemical exposures. The OELs mirror the outcome of the risk assessment and risk management performed by the standard setting actor. In paper I the OELs established by 18 different organisations or national regulatory agencies from the industrialised world were compared. The comparison concerned: (1) what chemicals have been selected and (2) the average level of exposure limits for all chemicals. In paper II the OELs established by 7 different national regulatory agencies of EU member states are compared to those of the European Commission (EC). In addition to the same comparisons as performed in the first study a comparison level was introduced (3) the similarity between the OELs of these EU member states and the OELs recommended by the EC.</p><p>List of OELs were collected through the web-pages of, and e-mail communication with the standard-setting agencies. The selection of agencies was determined by availability of the lists. The database of paper I contains OELs for a total of 1341 substances; of these 25 substances have OELs from all 18 organisations while more than one third of the substances are only regulated by one organisation alone. In paper II this database was narrowed down to the European perspective.  The average level of OELs differs substantially between organisations; the US OSHA exposure limits are (on average) nearly 40 % higher than those of Poland. Also within Europe there was a nearly as large difference. The average level of lists tends to decrease over time, although there are exceptions to this. The similarity index in paper II indicates that the exposure limits of EU member states are converging towards the European Commission’s recommended OELs. These two studies also showed that OELs for the same substance can vary significantly between different standard-setters. The work presented in paper III identifies steps in the risk assessment that could account for these differences. Substances for which the level of OELs vary by a factor of 100 or more were identified and their documentation sought for further scrutiny. Differences in the identification of the critical effect could explain the different level of the OELs for half of the substances. The results reported in paper III also confirm the tendency of older OELs generally being higher. Furthermore, several OELs were more than 30 years old and were based on out-dated knowledge. But the age of the data review could not account for all the differences in data selection, only one fifth of the documents referred to all available key studies. Also the evaluation of the key studies varied significantly.</p>

For the majority of the estimated 70,000 industrial chemical substances available on the European market today there is not enough information to enable a reasonably complete assessment of the risks that they might pose to man and the environment. Any strategy for the generation of additional data for these substances should aim at making testing as efficient as possible taking into account environmental and health protection, time, monetary cost and animal welfare. To achieve this, appropriate priority setting rules are needed. The main criterion currently used for regulatory priority setting for testing of industrial chemicals is production volume; the higher the production volume, the more information is required. This was also the main criterion in the former legislation, preceding REACH (Registration, Evaluation and Authorisation of Chemicals). The aim of this thesis is to evaluate other priority setting criteria and their implications for risk management, in particular classification and labelling. The first paper in this thesis includes a study of the efficiency ratio for some of the tests required for the notification of new substances, i.e. the ratio between the likelihood that the test will lead to a classification, and the monetary cost of performing the test. The efficiency ratio was determined for the standard tests for acute oral toxicity, irritation, sensitisation and subacute toxicity using data from 1409 new chemicals notified in Europe between 1994 and 2004. The results of this investigation suggest that, given limited resources for testing, it is more efficient to perform acute toxicity tests on a larger number of substances rather than to perform additional subacute toxicity studies on the substances already tested for acute toxicity. The second paper included in this thesis, reports the results from a comparative study of the bioaccumulating properties of substances being (a) classified as carcinogenic, mutagenic and/or toxic to reproduction (CMR-substances), or (b) classified as acutely toxic or (c) unclassified. The purpose of this investigation was to evaluate potential consequences of prioritising bioaccumulating chemicals for evaluation and testing, as this is one of the strategies prescribed in REACH. The results of this study suggest that bioaccumulating substances are neither over- nor underrepresented among the CMR-substances. This result lends support to the use of the bioconcentration factor for priority setting. The studies reported in this thesis utilize existing data on classification of substances as an indicator of the outcome of the risk assessment process, relating priority setting methods to the risk management measures that they give rise to. To the best of my knowledge there are still only very few studies published that address the issue of priority setting in chemicals control using this approach, and in my view there is need for more studies of priority setting methods and a further development of priority setting strategies that are science-based. / QC 20101115

In vitro genetic toxicity tests using cultured bacterial or mammalian cells provide a cost- and time-effective alternative to animal tests. Unfortunately, existing in vitro assays are not always reliable. This is in part due to the limited metabolic capacity of the cells used, which is often critical to accurately assess chemical genotoxicity. This limited metabolic capacity necessitates the use of exogenous sources of mammalian metabolic enzymes that can simulate in vivo mammalian metabolic activation reactions. In response to this, and other limitations, alongside the worldwide trend to reduce animal testing, there is an acute need to consider various strategies to improve in vitro mutagenicity assessment. This thesis first examined the utility of exogenous metabolic activation systems based on human hepatic S9, relative to conventional induced rat liver S9, for routine genetic toxicity assessment. This was accomplished by critically evaluating existing literature, as well as new experimental data. The results revealed the limitations of human liver S9 for assessment of chemical mutagenicity. More specifically, the analyses concluded that, due to the increased risk of false negative results, human liver S9 should not be used as a replacement for induced rat liver S9. To address the limitations of conventional mammalian cell genetic toxicity assays that require exogenous hepatic S9, the thesis next evaluated the utility of an in vitro mutagenicity assay based on metabolically-competent primary hepatocytes (PHs) derived from the transgenic MutaMouse. Cultured MutaMouse PHs were thoroughly characterized, and found to temporarily retain the phenotypic attributes of hepatocytes in vivo; they express hepatocyte-specific proteins, exhibit the karyotype of typical hepatocytes, and maintain metabolic activity for at least the first 24 hours after isolation. Preliminary validation of the in vitro MutaMouse PH gene mutation assay, using a panel of thirteen mutagenic and non-mutagenic chemicals, demonstrated excellent sensitivity and specificity. Moreover, inclusion of substances requiring a diverse array of metabolic activation pathways revealed comprehensive metabolic competence. Finally, the thesis further investigated the applicability domain of the in vitro MutaMouse PH assay by challenging the assay with selected azo compounds. Comparison of these results with those obtained using the in vivo MutaMouse TGR (transgenic rodent) assay revealed that MutaMouse PHs can carry out some forms of reductive metabolism. Overall, this thesis demonstrated that a gene mutation assay based on MutaMouse PHs holds great promise for routine assessments of chemical mutagenicity.

Occupational Exposure Limits (OELs) are used as an important regulatory instrument to protect workers’ health from adverse effects of chemical exposures. The main objective of this thesis is to study risk assessment practices in the setting of OEL in order to produce knowledge that will help improve the consistency and transparency of OELs. For the purpose of paper I a database of OELs for a total of 1341 substances was compiled. Of these, only 25 substances have OELs from all 18 included organisations while more than one third of the substances are only regulated by one organisation alone. The average level of OELs differs substantially between organisations; the US OSHA exposure limits are (on average) nearly 40 % higher than those of Poland. In paper II six EU member states’ OELs are compared to the European Commission’s OELs. Also within Europe there is a large difference concerning the average level of OELs (35%). The average level of lists tends to decrease over time, although there are exceptions to this. There are also indications that the exposure limits of EU member states are converging towards the European Commission’s OELs. The work presented in paper III identifies steps in the risk assessment that could account for the large differences in OELs for 14 different substances. Differences in the identification of the critical effect could explain the different level of the OELs for half of the substances. But the age of the data review could not account for all the differences in data selection, only one fifth of the documents referred to all available key studies. Also the evaluation of the key studies varied significantly. The aim of paper IV was to investigate how the Scientific Committee on Occupational Exposure Limits (SCOEL) of the European Commission uses assessment factors when proposing health-based indicative OELs. For only one third of the investigated OELs were explicit assessment factors given. On average the safety margin of the recommendations was 2.1 higher when an explicit assessment factor had been used. It is recommended that the SCOEL develop and adhere to a more articulate framework on the use of assessment factors. Paper V focuses on the Derived No-Effect Levels (DNELs) which are to be calculated under the new European Union REACH legislation. It is a comparison of the safety margins of 88 SCOEL recommendations with those of the corresponding worker-DNELs, derived according to the default approach as described in the REACH guidance document. Overall, the REACH safety margins were approximately six times higher than those derived from the SCOEL documentations but varied widely with REACH/SCOEL safety margin ratios ranging by two orders of magnitude, from 0.3 to 58. / QC 20110215

This thesis focuses on exacerbating chemicals risk in workplaces under the background of rapid industrialization in developing countries. The overall aim is to investigate the development of regulatory tools which aim at minimizing the health risks from chemical substances in the workplace. The contents of the thesis are divided into three sections: the profile of occupational diseases in China (paper I), occupational exposure limits (paper II and III), and comparison between chemicals regulat ions in Europe and China (paper IV). Paper I presents an analysis of the development of occupational diseases in China between 2000 and 2010. The number of recorded cases of occupational diseases increased rapidly in China during this period and the majority of cases were attributable to dust and other chemicals exposures. Difficulties in diagnosis and inefficient surveillance are major impediments to the proper identification and mitigation of occupational diseases. Migrant workers are extremely vulnerable to occupational hazards. Paper II investigates the state of harmonization of OELs between twenty-five OEL systems in Europe and Asia. The majority of the investigated organizations declare themselves to have been influenced by the American Conference of Governmental Industrial Hygienists (ACGIH), and in many cases this can be empirically confirmed. However, large international differences still exist in substance selection and in the level of OELs among organizations. Paper III explores the setting of risk-based OELs on non-threshold carcinogens. Relatively few agencies set risk-based OELs. Differences exist in policy, both regarding the magnitude of risk considered as tolerable or acceptable and whether a general risk level or case-by-case substance-specific risk levels are determined. In regards to the level of the OELs both differences in science and policy contribute, and it was not possible to determine which has the larger influence. Paper III explores the setting of risk-based OELs on non-threshold carcinogens. Relatively few agencies set risk-based OELs. Differences exist in policy, both regarding the magnitude of risk considered as tolerable or acceptable and whether a general risk level or case-by-case substance-specific risk levels are determined. In regards to the level of the OELs both differences in science and policy contribute, and it was not possible to determine which has the larger influence. Paper IV systematically compares the regulation systems for chemicals in the EU and China in terms of substances covered, requirement on information, risk assessment and risk management. It shows that the European and Chinese chemicals legislations are remarkably similar.The differences are larger in terms of substance coverage and data requirements than in terms of risk assessment and management. Substitution of hazardous substances is driven more by updates of the EU regulatory system than of the Chinese system. / <p>QC 20130830</p>

Health and environmental risks associated with emissions of hazardous chemicals from articles, including everyday consumer products such as clothes and toys, have become widely acknowledged internationally, particularly in the EU. This thesis contributes to new understandings of how these risks are currently managed within the EU and recommends actions for ensuring a safe and sustainable use of chemicals in articles. Paper I provides an overview and comparative analysis of regulatory strategies for managing risks of chemicals in articles in the EU. The in-depth analysis, which is focused on the Toys Safety Directive, the RoHS Directive, and REACH, shows that the legislations differ significantly. Differences include e.g. what criteria are used for the selection of substances to be targeted for regulation, and the kind of requirements and restrictions applied to the selected substances. It is concluded that product-specific directives are important complements to REACH in order to ensure a safe use of chemicals in articles. Paper II evaluates to what extent the regulation of chemicals in articles under REACH is coherent with the rules concerning chemicals in the Sewage Sludge Directive (SSD) and the Water Framework Directive (WFD). The results show that the majority of the chemicals that are prioritized for phase-out under the WFD or for concentration restrictions in sludge and soil under the SSD are allowed to be used in articles according to REACH. In order to avoid end-of-pipe problems and to increase resource efficiency, it is argued that it is necessary to minimize the input of chemicals identified as hazardous to health or the environment into articles. Paper III aims to clarify what the substitution principle means and how it can reasonably be applied as part of chemical policies. A general definition is proposed that gives equal weight to hazard, functionality and economical considerations, while at the same time recognizing that the aim of the substitution principle is to reduce hazards to human health and the environment. This paper also summarizes major methods to promote and implement the principle, discusses legislative approaches with regard to their ability to promote substitution of hazardous chemicals, and makes proposals for an efficient implementation of the principle. / <p>QC 20121119</p>

Occupational Exposure Limits (OELs) are used as an important regulatory instrument to protect workers’ health from adverse effects of chemical exposures. The OELs mirror the outcome of the risk assessment and risk management performed by the standard setting actor. In paper I the OELs established by 18 different organisations or national regulatory agencies from the industrialised world were compared. The comparison concerned: (1) what chemicals have been selected and (2) the average level of exposure limits for all chemicals. In paper II the OELs established by 7 different national regulatory agencies of EU member states are compared to those of the European Commission (EC). In addition to the same comparisons as performed in the first study a comparison level was introduced (3) the similarity between the OELs of these EU member states and the OELs recommended by the EC. List of OELs were collected through the web-pages of, and e-mail communication with the standard-setting agencies. The selection of agencies was determined by availability of the lists. The database of paper I contains OELs for a total of 1341 substances; of these 25 substances have OELs from all 18 organisations while more than one third of the substances are only regulated by one organisation alone. In paper II this database was narrowed down to the European perspective.  The average level of OELs differs substantially between organisations; the US OSHA exposure limits are (on average) nearly 40 % higher than those of Poland. Also within Europe there was a nearly as large difference. The average level of lists tends to decrease over time, although there are exceptions to this. The similarity index in paper II indicates that the exposure limits of EU member states are converging towards the European Commission’s recommended OELs. These two studies also showed that OELs for the same substance can vary significantly between different standard-setters. The work presented in paper III identifies steps in the risk assessment that could account for these differences. Substances for which the level of OELs vary by a factor of 100 or more were identified and their documentation sought for further scrutiny. Differences in the identification of the critical effect could explain the different level of the OELs for half of the substances. The results reported in paper III also confirm the tendency of older OELs generally being higher. Furthermore, several OELs were more than 30 years old and were based on out-dated knowledge. But the age of the data review could not account for all the differences in data selection, only one fifth of the documents referred to all available key studies. Also the evaluation of the key studies varied significantly.