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Haemolytic anaemia is often induced following prolonged exposure to chemical substances. Currently, under EU Council Directive 67/548/EEC, substances which induce such effects are classified as dangerous and assigned the risk phrase R48 'Danger of serious damage to health by prolonged exposure.' Whilst the general classification criteria for this endpoint are outlined in Annex VI of this Directive, they do not provide specific information to assess haemolytic anaemia. This review produced by the EU Working Group on Haemolytic Anaemia provides a toxicological assessment of haemolytic anaemia and proposes criteria that can be used in the assessment for classification of substances which induce such effects. An overview of the primary and secondary effects of haemolytic anaemia which can occur in rodent repeated dose toxicity studies is given. A detailed analysis of the toxicological significance of such effects is then performed and correlated with the general classification criteria used for this endpoint. This review intends to give guidance when carrying out an assessment for classification for this endpoint and to allow for better transparency in the decision-making process on when to classify based on the presence of haemolytic anaemia in repeated dose toxicity studies. The extended classification criteria for haemolytic anaemia outlined in this review were accepted by the EU Commission Working Group on the Classification and Labelling of Dangerous Substances in September 2004.

Two international ring studies were performed to develop appropriate parameters within standard toxicology study for screening of immunotoxicological potential of unknown substances. These studies followed OECD TG 407 and included a number of additional examinations. CSA was selected as model for its immunosuppressive and HCB as model for its immunostimulating effects. Reproducibility of data was defined by significant findings in at least 50% of participating laboratories. In-life clinical observations, values for WBC parameters, and changes of lymphoid organ weights suggested immune effects. Elevated IgM titers indicated increased antibody formation in HCB-exposed rats. Cellularity of T-cell compartments in thymus (medulla), spleen (PALS), and lymph nodes (paracortical zone of mesenteric and popliteal LN) were dose dependently decreased in CSA-treated rats. The numbers of follicular germinal centers were reduced in LN. HCB induced cellular proliferation in spleen marginal zones and endothelial activation in HEV of mesenteric and popliteal LN and GALT and in small pulmonary venules. Data obtained by specific immune parameters indicated immune effects; however, statistical inference was limited to low numbers of participating laboratories. In spleen, both substances decreased lymphoblast proliferation after ConA mitogen stimulation. Reduced numbers of antibody-forming cells in PFC assay indicated impaired T-cell-dependent humoral immunity by CSA, which was not seen for HCB. Altered fractions for B- and T-cell subpopulations were identified in spleen for both substances. In order to predict immunomodulatory effects of CSA or HCB, histomorphologic examination of lymphoid tissues resulted in the most reliable and sensitive data to distinguish immunosuppression and -stimulation.