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NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research

Toxicology and regulatory sciences bibliography and resources

Acute toxicity testing

Adoption of the Fixed Concentration Procedure (FCP) for acute inhalation studies

Sewell F et al, (2018). An evaluation of the fixed concentration procedure for assessment of acute inhalation toxicity. Regulatory Toxicology and Pharmacology 94: 22-32. doi: 10.1016/j.yrtph.2018.01.001

OECD (2017). Test no. 433: Acute Inhalation Toxicity: Fixed Concentration Procedure. OECD Publishing, Parisdoi: 10.1787/9789264284166-en

Sewell F et al, (2015). A global initiative to refine acute inhalation studies through the use of ‘evident toxicity’ as an endpoint: towards adoption of the fixed concentration procedure. Regulatory Toxicology and Pharmacology 73(3): 770-9. doi: 10.1016/j.yrtph.2015.10.018

Price C et al, (2011). A statistical evaluation of the effects of gender differences in assessment of acute inhalation toxicity. Human and Experimental Toxicology 30(3): 217-238. doi: 10.1177/0960327110370982

Stallard N et al, (2011). A new sighting study for the fixed concentration procedure to allow for gender differences. Human and Experimental Toxicology 30(3): 239-249. doi: 10.1177/0960327110370983

Applying the 3Rs in fish acute toxicity studies

Katsiadaki I et al, (2021). Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint. Ecotoxicology and Environmental Safety 223:112585. doi: 10.1016/j.ecoenv.2021.112585

Burden N et al, (2020). Key opportunities to replace, reduce and refine regulatory fish acute toxicity tests. Environmental Toxicology and Chemistry doi: 10.1002/etc.4824

Burden N et al, (2016). The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach. Regulatory Toxicology and Pharmacology 80:241-6. doi: 10.1016/j.yrtph.2016.05.032

Creton S et al, (2014). Application of the threshold approach for acute fish toxicity testing to plant protection products: a proposed framework. Chemosphere 96: 195-200. doi: 10.1016/j.chemosphere.2013.10.015

Redundancy in the acute toxicity testing of chemicals

Moore NP et al, (2013). Can acute dermal systemic toxicity tests be replaced with oral tests? A comparison of route-specific systemic toxicity and hazard classifications under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Regulatory Toxicology and Pharmacology 66(1): 30-7. doi: 10.1016/j.yrtph.2013.02.005

Creton S et al, (2010). Acute toxicity testing of chemicals – Opportunities to avoid redundant testing and use alternative approaches. Critical Reviews in Toxicology 40: 50-83. doi: 10.3109/10408440903401511

Seidle T et al, (2010). Cross-sector review of drivers and available 3Rs approaches for acute systemic toxicity testing. Toxicological Sciences 116(2): 382-396. doi: 10.1093/toxsci/kfq143

Single dose acute toxicity studies for new medicines

Chapman K et al, (2010). The value of acute toxicity studies to support the clinical management of overdose and poisoning: a cross-discipline consensus. Regulatory Toxicology and Pharmacology 58(3): 354-359. doi: 10.1016/j.yrtph.2010.07.003

Robinson S and Chapman K (2009). Are acute toxicity studies required to support overdose for new medicines? Regulatory Toxicology and Pharmacology 55(1): 110. doi: 10.1016/j.yrtph.2009.06.012

Robinson S et al, (2008). A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development. Regulatory Toxicology and Pharmacology 50(3): 345-352. doi: 10.1016/j.yrtph.2007.11.009

Chapman K and Robinson S (2007). Workshop Report: Challenging the regulatory requirement for acute toxicity studies in the development of new medicines. NC3Rs, London


Animals in environmental safety testing

Applying the one concentration approach in fish bioaccumulation studies

Burden N et al, (2017). Reducing the number of fish in regulatory bioconcentration testing: Identifying and overcoming the barriers to using the 1-concentration approach. Integrated Environmental Assessment and Management 13(1): 212-4. doi: 10.1002/ieam.1851

Creton S et al, (2013). Reducing the number of fish in bioconcentration studies for plant protection products by reducing the number of test concentrations. Chemosphere 90(3): 1300-4. doi: 10.1016/j.chemosphere.2012.09.029

Applying the 3Rs in non-mammalian endocrine disruptor testing

Ortego L et al, (2021). The Extended Amphibian Metamorphosis Assay (EAMA): A thyroid-specific and less animal-intensive alternative to the Larval Amphibian Growth and Development Assay (LAGDA, OECD TG 241). Environmental Toxicology and Chemistry 40(8):2135-2144.  doi: 10.1002/etc.5078

Burden N et al, (2021). Investigating endocrine disrupting properties of chemicals in fish and amphibians: opportunities to apply the 3Rs. Integrated Environmental Assessment and Management doi: 10.1002/ieam.4497

Wheeler JR et al, (2021). Hormone data collection in support of endocrine disruption (ED) assessment for aquatic vertebrates: Pragmatic and animal welfare considerations. Environ International 146:106287. doi: 10.1016/j.envint.2020.106287

Lagadic L et al, (2019). Recommendations for reducing the use of fish and amphibians in endocrine-disruption testing of biocides and plant protection products in Europe. Integrated Environmental Assessment and Management 15: 659-662. doi: 10.1002/ieam.4156

Assessing the need for chronic fish studies on formulated pesticides

Creton S et al, (2010). Challenging the requirement for chronic fish toxicity studies on formulated plant protection products. Toxicology Letters 199(2): 111-4. doi: 10.1016/j.toxlet.2010.08.019

Reducing repetition of regulatory vertebrate ecotoxicology studies

Burden N et al, (2017). Reducing repetition of regulatory vertebrate ecotoxicology studies. Integrated Environmental Assessment and Management 13(5) 955-7. doi: 10.1002/ieam.1934

Reviews / Other / Miscellaneous

Burden N et al, (2016). Advancing the 3Rs in regulatory ecotoxicology: A pragmatic cross-sector approach. Integrated Environmental Assessment and Management 12(3): 417-421. doi: 10.1002/ieam.1703

Lillicrap A et al, (2016). Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status. Environmental Toxicology and Chemistry 35(11): 2637-2646. doi: 10.1002/etc.3603

Hutchinson TH et al, (2015). Promoting the 3Rs to enhance the OECD fish toxicity testing framework. Regulatory Toxicology and Pharmacology 76: 231-3. doi: 10.1016/j.yrtph.2016.02.006

Burden N and Hutchinson TH (2015). Benefits of the ARRIVE Guidelines for improving scientific reporting in ecotoxicology – An academic perspective. Environmental Toxicology and Chemistry 34(11): 2446-8. doi: 10.1002/etc.3111


Exposure and dose selection

Applying exposure science to increase the utility of non-animal data in efficacy and safety testing

Sewell F et al, (2017). The current status of exposure-driven approaches for chemical safety assessment: A cross-sector perspective. Toxicology 389: 109-117. doi: 10.1016/j.tox.2017.07.018

NC3Rs/Unilever (2017). Workshop report: Applying exposure science to increase the utility of non-animal data in efficacy and safety testingNC3Rs/Unilever, London.  

Rowbotham AL and Gibson RM (2011). Exposure-driven risk assessment: Applying exposure-based waiving of toxicity tests under REACH. Food and Chemical Toxicology 49(8): 1661-1673. doi: 10.1016/j.fct.2011.03.050

Pharmacokinetics in candidate selection

Beaumont K et al, (2011). Towards an integrated human clearance prediction strategy that minimizes animal use. Journal of Pharmaceutical Sciences 100:1167–1783. doi: 10.1002/jps.22635

Lave T et al, (2009). Human clearance prediction: shifting the paradigm. Expert Opinion on Drug Metabolism & Toxicology 5(9): 1039-1048. doi: 10.1517/17425250903099649

Refining MTD studies

ECETOC Guidance on Dose Selection (2020). Technical report 138, Brussels, March 2021. ISSN-2079-1526-138.

Chapman K et al, (2013). A global pharmaceutical company initiative: An evidence-based approach to define the upper limit of body weight loss in short term toxicity studies. Regulatory Toxicology and Pharmacology 67(1): 27-38. doi: 10.1016/j.yrtph.2013.04.003

Robinson S et al, (2009). Guidance on dose level selection for regulatory general toxicology studies for pharmaceuticals. NC3Rs/LASA, London.

Toxicokinetics in the chemicals industry

Tan YM et al, (2021). Opportunities and challenges related to saturation of toxicokinetic processes: implications for risk assessment. Regulatory Toxicology and Pharmacology 127: 105070. doi: 10.1016/j.yrtph.2021.105070

Sewell F et al, (2020). Use of the kinetically-derived maximum dose: Opportunities for delivering 3Rs benefits. Regulatory Toxicology and Pharmacology. 116:104734. doi: 10.1016/j.yrtph.2020.104734

Creton S et al, (2012). Use of toxicokinetics to support chemical evaluation: Informing high dose selection and study interpretation. Regulatory Toxicology and Pharmacology 62(2): 241-7. doi: 10.1016/j.yrtph.2011.12.005

Creton S et al, (2009). Application of toxicokinetics to improve chemical risk assessment: Implications for the use of animals. Regulatory Toxicology and Pharmacology 55: 291-9. doi: 10.1016/j.yrtph.2009.08.001


General reviews

Burden N et al, (2015). Pioneering better science through the 3Rs: An introduction to the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs). Journal of the American Association for Laboratory Animal Science 54(2): 198-208. 

Holmes AM et al, (2010). Working in partnership to advance the 3Rs in toxicity testing. Toxicology 267(1-3): 14-9. doi: 10.1016/j.tox.2009.11.006


New approach methodologies (NAMs) in toxicology

Application of non-animal approaches for decision making in chemical safety assessment

Sewell F et al, (2021). Rethinking Agrochemical Safety Assessment: A Perspective. Regulatory Toxicology and Pharmacology 127:105068. doi: 10.1016/j.yrtph.2021.105068

LaLone C et al, (2021). International consortium to advance cross species extrapolation of the effects of chemicals in regulatory toxicology. Environmental Toxicology and Chemistry. 126: e105029. doi: 10.1002/etc.5214

Prior H et al, (2019). Reflections on the progress towards non-animal methods for acute toxicity testing of chemicals. Regulatory Toxicology and Pharmacology 102 30-3. doi: 10.1016/j.yrtph.2018.12.008

Hoffmann S et al, (2018). Non-animal methods to predict skin sensitization (I): the Cosmetics Europe database. Critical Reviews in Toxicology 48: 344-358. doi: 10.1080/10408444.2018.1429385

Kleinstreuer NC et al, (2018). Non-animal methods to predict skin sensitization (II): an assessment of defined approaches. Critical Reviews in Toxicology 48: 359-374. doi: 10.1080/10408444.2018.1429386

van Vliet E et al, (2018). State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop. ALTEX 35: 179-192. doi: 10.14573/altex.1709011

Sewell F et al, (2017). Steps towards the international regulatory acceptance of non-animal methodology in safety assessment. Regulatory Toxicology and Pharmacology 89: 50-6. doi: 10.1016/j.yrtph.2017.07.001

Burden N et al, (2015). Testing chemical safety: What is needed to ensure the widespread application of non-animal approaches? PLoS Biology 13(5): e1002156. doi: 10.1371/journal.pbio.1002156

Burden N et al, (2015). Aligning the 3Rs with new paradigms in the safety assessment of chemicals. Toxicology 330: 62-6. doi: 10.1016/j.tox.2015.01.014

Adler S et al, (2011). Alternative (non-animal) methods for cosmetics testing: current status and future prospects – 2010. Archives of Toxicology 85(5): 367-485. doi: 10.1007/s00204-011-0693-2

In vitro approaches for carcinogenicity testing

Luijten M et al, (2020). A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals. Regulatory Toxicology and Pharmacology. 118: 104789. doi: 10.1016/j.yrtph.2020.104789

Creton S et al, (2012). Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs. Mutagenesis 27(1): 93-101. doi: 10.1093/mutage/ger053

Modelling and computational approaches

Gellatly N and Sewell F (2019). Regulatory acceptance of in silico approaches for the safety assessment of cosmetic-related substances. Computational Toxicology 11: 82-9.  doi: 10.1016/j.comtox.2019.03.003

Hasselgren C et al, (2019). Genetic toxicology in silico protocol. Regulatory Toxicology and Pharmacology, 107: 104403. doi: 10.1016/j.yrtph.2019.104403

Myatt GJ et al, (2018). In silico toxicology protocols. Regulatory Toxicology and Pharmacology 96: 1-17. doi: 10.1016/j.yrtph.2018.04.014

Pathways based approaches

Rivetti C et al, (2020). Vision of a near future: Bridging the human health-environment divide. Toward an integrated strategy to understand mechanisms across species for chemical safety assessment. Toxicology In Vitro 62: 104692. doi: 10.1016/j.tiv.2019.104692

Sewell F et al, (2018). The future trajectory of adverse outcome pathways: a commentary. Archives of Toxicology 92(4): 1657-1661. doi: 10.1007/s00204-018-2183-2

Burden N et al, (2015). Adverse Outcome Pathways can drive non-animal approaches for safety assessment. Journal of Applied Toxicology 35(9): 971-975. doi: 10.1002/jat.3165

Reducing animal use in the safety assessment of nanomaterials

Burden N et al,(2021). Opportunities and Challenges for Integrating New In Vitro Methodologies in Hazard Testing and Risk Assessment. Small. (15):e2006298. doi: 10.1002/smll.202006298

Burden N et al, (2017). The 3Rs as a framework to support a 21st century approach for nanosafety assessment. Nano Today 12: 10-13. doi: 10.1016/j.nantod.2016.06.007

Burden N et al, (2017). Aligning nanotoxicology with the 3Rs: What is needed to realise the short, medium and long-term opportunities? Regulatory Toxicology and Pharmacology 91: 257-266. doi: 10.1016/j.yrtph.2017.10.021

Use of human or engineered tissues in safety assessment

Jackson SJ et al, (2018). The use of human tissue in safety assessment. Journal of Pharmacological and Toxicological Methods 93:29-34. doi: 10.1016/j.vascn.2018.05.003

Holmes A et al, (2017). Rising to the challenge: applying biofabrication approaches for better drug and chemical product development. Biofabrication 9: 033001. doi: 10.1088/1758-5090/aa7bbd

Holmes A et al, (2015). Assessing drug safety in human tissues — what are the barriers? Nature Reviews Drug Discovery 14: 585–7 (2015). doi: 10.1038/nrd4662

Westmoreland C and Holmes A (2009). Assuring consumer safety without animals: Applications for tissue engineering. Organogenesis 5: 67-72. doi: 10.4161/org.5.2.9128

Holmes A et al, (2009). Engineering tissue alternatives to animals: applying tissue engineering to basic research and safety testing. Regenerative Medicine 4: 572-92.


Safety pharmacology

Abuse potential studies

O'Connor E et al, (2011). The predictive validity of the rat self-administration model for abuse liability. Neuroscience & Biobehavioral Reviews 35(3): 912-938. doi: 10.1016/j.neubiorev.2010.10.012

Assessing the predictive value of safety pharmacology studies

Jackson SJ et al, (2019). Neurofunctional test batteries in safety pharmacology – Current and emerging considerations for the drug development process. Journal of Pharmacological and Toxicological Methods 100: 106602 doi: 10.1016/j.vascn.2019.106602

Redfern WS et al, (2019). The functional observational battery and modified Irwin test as global neurobehavioral assessments in the rat: Pharmacological validation data and a comparison of methods. Journal of Pharmacological and Toxicological Methods 98: 106591 doi: 10.1016/j.vascn.2019.106591

Mead AN et al, (2016). Assessing the predictive value of the rodent neurofunctional assessment for commonly reported adverse events in phase I clinical trials. Regulatory Toxicology and Pharmacology. 80: 348-357. doi: 10.1016/j.yrtph.2016.05.002

Social housing during rodent and non-rodent telemetry recordings

Prior H and Holbrook M (2021). Strategies to encourage the adoption of social housing during cardiovascular telemetry recordings in non-rodents. Journal of Pharmacological and Toxicological Methods 108: 106959. doi: 10.1016/j.vascn.2021.106959

Skinner M et al, (2019). Social-housing and use of double-decker cages in rat telemetry studies. Journal of Pharmacological and Toxicological Methods 96: 87-94. doi: 10.1016/j.vascn.2019.02.005

Prior H et al, (2016). Social housing of non-rodents during cardiovascular recordings in safety pharmacology and toxicology studies. Journal of Pharmacological and Toxicological Methods 81: 75-87. doi: 10.1016/j.vascn.2016.03.004

Reviews / Other

Guns PJ et al, (2020).  INSPIRE: A European training network to foster research and training in cardiovascular safety pharmacology.  Journal of Pharmacological and Toxicological Methods 105: 106889. doi: 10.1016/j.vascn.2020.106889

Saleem U et al, (2020). Blinded, Multicenter Evaluation of Drug-induced Changes in Contractility Using Human-induced Pluripotent Stem Cell-derived Cardiomyocytes. Toxicological Sciences 176: 103–123. doi: 10.1093/toxsci/kfaa058

Tse K et al, (2018). Pharmacological validation of individual animal locomotion, temperature and behavioural analysis in group-housed rats using a novel automated home cage analysis system: A comparison with the modified Irwin test. Journal of Pharmacological and Toxicological Methods 94: 1-13. 

Grant C et al, (2017). Provision of food and water in rodent whole body plethysmography safety pharmacology respiratory studies - Impact on animal welfare and data quality. Journal of Pharmacological and Toxicological Methods. 88: 79-84. doi: 10.1016/j.vascn.2017.07.004

Redfern W et al, (2017). Automated recording of home cage activity and temperature of individual rats housed in social groups: The Rodent Big Brother. PLoS One 12(9): e0181068. doi: 10.1371/journal.pone.0181068




Study designs for pharmaceutical and chemical development

Maximising the success of bile duct cannulation (BDC) studies

Burden N et al, (2017). Maximizing the success of bile duct cannulation studies in rats: recommendations for best practice. Laboratory Animals 51(5): 457-464. doi: 10.1177/0023677217698001.


Coleman D et al, (2017). Capillary microsampling in nonclinical safety assessment: practical sampling and bioanalysis from a CRO perspective.  Bioanalysis 9(10): 787-798. doi: 10.4155/bio-2017-0032

Chapman K et al, (2014). Overcoming the barriers to the uptake of nonclinical microsampling in regulatory safety studies. Drug Discovery Today 19(5): 528-532. doi: 10.1016/j.drudis.2014.01.002.

Chapman K et al, (2014). Reducing pre-clinical blood volumes for toxicokinetics: toxicologists, pathologists and bioanalysts unite. Bioanalysis 6(22): 2965-8. doi: 10.4155/bio.14.204.

Reducing animal use in monoclonal antibody (mAb) development

Sewell F et al, (2017). Challenges and opportunities for the future use of monoclonal antibody development: improving safety assessment and reducing animal use. mAbs 9(5): 742-755. doi: 10.1080/19420862.2017.1324376.

Chapman K et al, (2016). Waiving in vivo studies for monoclonal antibody biosimilar development: National and global challenges. mAbs 8(3): 427-435. doi: 10.1080/19420862.2016.1145331.

Chapman KL et al, (2012). The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates. Regulatory Toxicology and Pharmacology 62(2): 347-354. doi: 10.1016/j.yrtph.2011.10.016.

Buckley LA et al, (2011). Considerations regarding nonhuman primate use in safety assessment of biopharmaceuticals. International Journal of Toxicology 30(5): 583-590. doi: 10.1177/1091581811415875.

Chapman KL et al, (2010). The future of non-human primate use in mAb development. Drug Discovery Today 15(5-6): 235-242. doi: 10.1016/j.drudis.2010.01.002.

Chapman K et al, (2009). Preclinical development of monoclonal antibodies: considerations for the use of non-human primates. mAbs 1(5): 505-516. doi: 10.4161/mabs.1.5.9676.

Chapman K et al, (2007). Preclinical safety testing of monoclonal antibodies: the significance of species relevance. Nature Reviews Drug Discovery 6(2): 120-6. doi: 10.1038/nrd2242.

NC3Rs/ABPI (2006). Workshop report: Opportunities for reducing the use of non-human primates in the development of monoclonal antibodiesNC3Rs/ABPI, London.

Reducing the use of recovery animals

Sewell F et al, (2014). Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials. Regulatory Toxicology and Pharmacology 70(1): 413-429. doi: 10.1016/j.yrtph.2014.07.018.

Review of the use of two species

Namdari R et al, (2021). Species selection for nonclinical safety assessment of drug candidates: Examples of current industry practice. Regulatory Toxicology and Pharmacology 126: 105029. doi: 10.1016/j.yrtph.2021.105029.

Prior H et al, (2020). Justification for species selection for pharmaceutical toxicity studies. Toxicology Research 9: 758-770. doi: 10.1093/toxres/tfaa081.

Prior H et al, (2020). Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation. Regulatory Toxicology and Pharmacology 113: 104624. doi: 10.1016/j.yrtph.2020.104624.

Prior H et al, (2019). Integration of consortia recommendations for justification of animal use within current and future drug development paradigms. International Journal of Toxicology 38(4), 319–325. doi: 10.1177/1091581819852922.

Prior H et al, (2018). Reviewing the utility of two species in general toxicology related to drug development. International Journal of Toxicology 37(2): 121-4. doi: 10.1177/1091581818760564.

Reviews / Other

Prior H et al, (2021). Refining Procedures within Regulatory Toxicology Studies: Improving Animal Welfare and Data. Animals 11: 3057. doi: 10.3390/ani11113057.

Sewell F et al, (2021). Preclinical screening for antidepressant activity – shifting focus away from the Forced Swim Test to the use of translational biomarkers. Regulatory Toxicology and Pharmacology 125:105002. doi: 10.1016/j.yrtph.2021.105002.

Lilley E et al, (2021). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals. Biologicals 74: 24-27. doi: 10.1016/j.biologicals.2021.10.002

Clements JM et al, (2020). Predicting the Safety of Medicines in Pregnancy: a Workshop Report. Reproductive Toxicology, S0890-6238(20)30024-1. doi: 10.1016/j.reprotox.2020.02.011.

NC3Rs (2019). Workshop report: Applying the 3Rs in pharma: Improving delivery of innovative medicines to patients. NC3Rs, London.

Prior H et al, (2018). Overview of 3Rs opportunities in drug discovery and development using non-human primates. Drug Discovery Today: Disease Models 23, 11-16. doi: 10.1016/j.ddmod.2017.11.005.

Sewell F et al, (2016). Opportunities to apply the 3Rs in safety assessment programs. Institute for Laboratory Animal Research Journal 57(2): 234-245. doi: 10.1093/ilar/ilw024.

Chapman KL et al, (2013). Pharmaceutical toxicology: designing studies to reduce animal use, while maximizing human translation. Regulatory Toxicology and Pharmacology 66(1): 88-103. doi: 10.1016/j.yrtph.2013.03.001.

Sparrow SS et al, (2011). Opportunities to minimise animal use in pharmaceutical regulatory general toxicology: A cross company review. Regulatory Toxicology and Pharmacology 61(2): 222-9. doi: 10.1016/j.yrtph.2011.08.001.