Lecture 11: Neurotoxicity

What is the Broad Definition of Neurotoxicity?

• Any adverse effect on the structure or function of the central and/or peripheral nervous system by a biological, chemical or physical agent

• These effects may be permanent or reversible, produced by a neuropharmacological or neurodegenerative property of a neurotoxicant or the result of a direct or indirect action on the nervous system

  • Defined by the US Interagency Committee on Neurotoxicology

How is Neurotoxicity Defined by Pharma?

• It is narrowly defined as exposure to a naturally occurring or man-made substance that causes damage to nervous tissue → it produces irreversible changes

• Definition doesn’t include reversible, pharmacological, receptor-mediated effects of centrally acting drugs

• It includes agents defined as neurotoxins or neurotoxicants

Why is Neurotoxicity More of A Concern?

• Irreversible damage to neurons is serious, as unlike intestinal epithelial cells, which have a 5-day turnover, the vast majority of neurons in the adult brain are there for the duration of an individual’s lifetime

• Finite supply → can’t afford to lose any

What is the Neurotoxicity Continuum?

• Monitors the progression of nervous system toxicity:

• Functional effects

  • Pharmacodynamic or receptor-mediated responses

  • Time-course related to the PK of drug response

  • Effects fully reversible

• Adaptive changes (occurs following repeated dosing)

  • Altered gene expression

  • Epigenetic changes

  • Receptor up/downregulation

  • Altered neurochemistry

  • Effects may persist after drug removal from body

• Structural changes

  • changes in synaptic plasticity

  • Inhibition of neurogenesis

  • Long-term or permanent

• Neurodegeneration

  • Loss of neurons and/ or glial cells

  • Permanent damage and changes in nervous system structure & function

How does reversibility change across the neurotoxicity continuum?

• Functional effects → Fully reversible

• Adaptive changes → May persist after drug removal

• Structural changes → Long-term/permanent

• Neurodegeneration → Permanent

Do all drugs progress along the neurotoxicity continuum?

• Some drugs begin with functional effects and progress with higher doses or repeated exposure

• Most drugs that cause functional effects do not progress beyond causing adaptive changes

How Does Neurotoxicity Differ Across the Body?

• Neurotoxicity varies in different parts of the central and peripheral nervous system

• This includes:

  • Central nervous system neurotoxicity

  • Peripheral neuropathy

  • Retinal degeneration

  • Optic nerve degeneration

  • Ototoxicity (toxicity to the auditory apparatus or auditory neuronal pathways)

  • Impairment of other special senses

  • Developmental neurotoxicity

What Chemicals Cause Neurotoxicity?

• Pharmaceuticals

• Drugs of abuse

• Organic solvents

• Heavy metals

• Pesticides

• Naturally occurring neurotoxins

• Gases

• Chemical warfare agents

• Research tools

What are common functional drug-induced neurotoxic effects?

• These effects are typically related to the pharmacodynamics of the drugs, with their time course reflecting the pharmacokinetics

• CNS effects:

  • Fatigue, somnolence, insomnia

  • Cognitive impairment, disorientation

  • Anxiety, depression, personality changes

  • Hallucinations, suicidal ideation

• Motor effects:

  • Tremor

  • Motor incoordination

  • Involuntary movements

  • Seizures

• Sensory effects:

  • Dizziness

  • Visual dysfunction

  • Auditory dysfunction

  • Paraesthesia

• Autonomic/other effects:

  • Nausea

  • Sexual dysfunction

  • Anorexia / hyperphagia

  • Abuse/dependence liability

What are common structural drug-induced neurotoxic effects?

• Central nervous system neurotoxicity

• Peripheral neuropathy (sensory and/or motor)

• Retinal degeneration

• Optic nerve degeneration

• Ototoxicity

What is an early example of functional drug-induced neurotoxicity?

• 1676, Virginia — ingestion of Jimson Weed (used in salad) → example of functional adverse effects on the nervous system

• Contains: Atropine, Hyoscyamine, Scopolamine

• These are muscarinic antagonists that can enter the brain

• Ingestion at high doses causes:

  • Delirium

  • Hallucinations

  • Amnesia

Do Neruons Exist in Isolation?

• No:

  • They are suppourted by glial cells → provide structural and biochemical/nutritional support

  • Neurons communicate with each other largely via specialised junctions termed synapses

• The synapse is the main site of action for acute adverse functional effects, with neurotoxicity involving any aspect of neuronal function, e.g. myelin sheath and axonal transport, as well as synaptic transmission.

What is the Main Site of Action for Acute Adverse Functional Effects

• The synapse

• Neurotoxicity can involve any aspect of neuronal function e.g., myelin sheath, axonal transport, as well as synaptic transmission

What are the Direct Mechanism of drug-induced neurotoxicity?

• Disruption of mitochondrial function

• Oxygen free radical formation

• Release of excitatory amino acids

• Ion channel inhibition

• Apoptosis

• Selective neurotransmitter depletion

• Interruption of axonal transport

What are the Indirect Mechanisms of drug-induced neurotoxicity?

• Hypoglycaemia

• Hypoxia

• Ischaemia

• Disruption of the blood-brain barrier

• Hepatotoxicity

• Vitamin deficiency (incl. B₆; folic acid; B₁₂; riboflavin)

• Coagulation disorders

• Renal failure

• Electrolyte disorders

• Endocrine disorders

  • These do not have to enter the brain to cause CNS toxicity

What Risk Factors Increase Susceptibility to Drug Induced Neurotoxicity?

• Individual risk factors: can promote or exacerbate drug-induced neurotoxicity, e.g.

  • pharmacogenetic differences;

  • ageing; history of neurological disorders;

  • compromised brain function;

  • Also, anything listed under ‘indirect’ caused by drug co-therapy or disease.

• Drug-drug interactions

  • e.g. cocaine + alcohol → undergo a (rare) chemical reaction to produce a neurotoxin

Why is the Brain Generally Succeptible to Oxidative Stress?

• High content of polyunsaturated fatty acids

• Low level of antioxidants

• Presence of transition metals

• High levels of oxygen consumption

Why are Dopaminergic Neurons In Particular susceptible to Oxidative Stress?

• Dopamine itself is a ‘neurotoxic time bomb’: it is readily oxidised, causing oxidative stress.

• Monoamine Oxidase (MAO) catalyses the deamination of dopamine, with hydrogen peroxide as a by-product.

• Dopamine auto-oxidation produces the superoxide anion (O₂^-) and hydrogen peroxide.

• Therefore, any drugs increasing release of dopamine can be neurotoxic to dopaminergic neurones by this mechanism.

Why are Dopaminergic Neurons in the Substantia Nigra Vulnerable to Oxidative Stress?

• Role: Motor function

• Reason for higher risk: Dopamine metabolism produces ROS

• Dopamine is both a neurotransmitter and a neurotoxin

• Effects of dysfunction/degeneration: Parkisons’s

Why are Retinal Pigment Epithelial Cells Vulnerable to Oxidative Stress?

• Role: (Physical) Support/Nutritional support (etc.) to photoreceptors

• Effects of dysfunction/degeneration: Loss of vision

• Reason for higher risk: Exposure to high levels of UV light and oxygen from high blood supply can damage the cells

What are the key features of CNS neurotoxicity?

• Can arise from various drug classes (including drugs of abuse)

• Different brain regions can be affected depending on the drug

• Symptoms depend on the brain region damaged

What are the key features of Peripheral Neuropathy?

• A common side effect of certain drug classes

  • e.g., Anti-microbials, oncology/anti-neoplastic agents, (some) cardiovascular drugs, some CNS drugs (+Micellanious)

• Initial symptoms: numbness/ tingling/ pins and needles

• Symptoms may progress to loss of function

• Can be subdivided into:

  • Neuronapathies (affect cell bodies)

  • Myelinopathies

  • Distalaxonopathies

• Different drugs are specific in the effects they cause → some drugs have multiple sites of actions

What are the key features of Retinal Damage/ Degeneration?

• A large number of drugs that cover a range of therapeutic classes are associated with retinal damage or degeneration

• Some of these drugs have a stronger association with producing this damage than others

What are the key features of Optic Nerve Degeneration?

• Some drugs cause optic nerve damage

• Some of these associations with drug and nerve damage are stronger than others

• As such, incidence may be low but still cause damage in some individuals

What are the key features of Ototoxicity?

• Drugs associated with damage to the cochlea

• No current screen for ototoxicity in drug development (not required by pharma) → ethical obligation to identify drugs that are potentially damaging to hearing (Chu et al, 2008)

• Functional testing is possible but not required

• Histopathology is difficult → sectioning requires the bone to be dissolved to access the soft tissue

• Some marketed drugs may be occult ototoxins → lurk undetected until drug reaches the market

• E.g. antibiotics (Gentamycin) and cisplatin agents can cause hearing loss

What Approaches are Avaliable to Detect and Assess Drug Induced Neurotoxicity Pre-Clinically?

• In silico:

  • Machine learning (Quantitative Structure Toxicity Relationship (QSTR)

• In vitro:

  • Neuronal cultures

  • In vitro electrophysiology (ion channels; neurons, slices, MEAs)

• In vivo:

  • Behavioural/ neurological assessments

  • Neurophysiological recordings, e.g. EEG, ERG, EMG, ABR/BAER, Nerve conduction velocity

  • Neurochemical, e.g. in vivo microdialysis, soluble biomarkers

  • Neuroimaging, e.g. MRI, MRS, PET, SPECT

• Post mortem:

  • Neurohistopathology (incl. immunohistochemistry, GFP)

How is the FDA Aiming to Replacing Animal Studies?

• In 2025, the FDA (US) issued a roadmap to replace animal studies with novel alternative methods

  • This includes in silico and in vitro methods

  • This is an aggressive timeline

• Questioned by many as to whether this will be achieved

• Development observed in in vitro neurotoxicity pathways recently → highlights the potential for this change.

What is the In Silico Neurotoxicity Assessment for Peripheral Neuropathy?

• Use of machine learning models and training sets of drugs

• A training set library of 95 drugs was created, including approved drugs with varying risks of peripheral neuropathy

• Using 60 of these drugs, they were able to predict with a reasonable degree of certainty from the molecular structure whether they would cause peripheral neuropathy

  • The outcome of these drugs was known but not built into the model

• This is an example of QSTR (Quantitative Structure Toxicity Relationships)

What methods are used in in vitro neurotoxicity assessment?

• Significant progress made in the last 15 years

• Various approaches present and can be combined, e.g. ‘on-a-chip’; multi-electrode arrays

• Functional readouts:

  • Electrophysiological recordings

  • Live cell Ca2+ imaging

  • Live cell fluorescent imaging

  • Neurotransmitter release

• Morphological readouts:

  • Dendritic spine morphology and density

  • Neurite outgrowth and synapse density

  • Cell viability

  • (Soluble) Biomarkers of neurotoxicity

• Also: knockdown receptor targets to investigate the mechanism of action of drug induced neurotoxicity in in vitro assessments

What does the early in vitro assay using rat hippocampal neurons show about chemotherapy-induced neurotoxicity?

• In Rat primary hippocampal neurons taxol, methotrexate, cisplatin (anti-cancer agents) were tested

• Findings:

  • Disruption/loss of dendritic processes

  • Loss of microfilaments

• Shown using markers MAP2 (red), a dendrite specific marker and Actin (green) → microfilament protein

• Example of early in vitro neurotoxicity assay showing chemotherapy-induced neuronal damage

What does the in vitro assay using mouse primary cerebellar granule neurons show about Beta-Bungratoxin Induced neurotoxicity?

• High content imaging, analysing neurite outgrowth

• Revealed reduced neurite outgrowth in the presence of the toxin

  • Used as a biomarker of neurotoxicity

What does the in vitro assay using hiPSC-Derived Neurons and Glia Show in Neurotoxicity Assessments of MethylMecury ?

• In hiPSC-Derived Neurons and Glia, the heavy metal neurotoxin, methylmercury, was tested

• Findings: Inhibition of neurite outgrowth by the neurotoxin

  • Dose/concentration-related effect and a decrease in neurite outgrowth were observed

What are key features of hiPSC neurone-glia in vitro neurotoxicity assessments?

• Shift from mouse/rat cells to human stem cells since ~2016

• Use of human iPSC-derived neuron-astrocyte 3D cultures to profile neurotoxicants

• High-throughput functional readout: spontaneous Ca²⁺ oscillation

What did human stem cell–derived neurons reveal about different neurotoxins?

• Different neurotoxins produced distinct functional effects

• Each neurotoxin generated a unique Ca²⁺ oscillation signature

• Demonstrates ability to profile pharmacological agents using human stem cell–derived neurons

What are 3D brain organoids in in vitro neurotoxicity assessment?

• Self-assembled 3D structures generated from various neural and neuronal subtypes that resemble the human embryonic brain, owing to their spatial organisation and the ability to replicate gene expression in vitro

• Consists of various neurons and glial cells

• Used to study neurotoxicity in vitro

What is guided differentiation in 3D brain organoids?

• The development of brain region-specific organoids using growth factors

• Creates distinct brain region architecture and composition

• Allows the investigation into region-specific neurotoxicity in response to neurotoxic drugs in vitro by assessing

  • Morphological responses

  • Neurochemical responses

  • Electrophysiological responses

What are 3D brain organoids “on a chip” in in vitro neurotoxicity assessment?

• Cells supported in a microfluidic device

• Allows culture medium to flow and perfuse, and for drug application (Rather than tissue present in a static fluid well)

• Different types of microfluidic devices exist → all currently in the validation phase

• Different groups are using different types of microfluidic devices

• There has been an explosion of this in recent years in different organ systems, including the brain

What are 3D retinal organoids in in vitro neurotoxicity assessment?

• Involves the reproduction of the retina’s structure in vitro

• Movement from a simple 2D cell type previously used in vitro for ~40 years to sophisticated in vitro systems

• Movement from animal-derived cells to human-induced stem cell-based preparations

What is the Peripheral Nerve On-A-Chip in in vitro neurotoxicity assessment?

• Involves a microfluidic system

• Primary tissue placed on one part of the device

• Long channel present to allow for axon growth

• Recordings of conduction velocity and microscopic imaging is possible

What does Bortezomib show in a Peripheral Nerve On-A-Chip in in vitro neurotoxicity assessment?

• A drug that causes peripheral neuropathy

• Low concentrations cause a decrease in nerve conduction rather than a decrease in cell viability

• Demonstrates the importance of measuring both functional and morphological imaging type outcomes

How are In Vitro Hippocampal Slice Preperations Used to Investigate Seizure Liability?

• Rat hippocampal slice procedure used for many years (~20yrs) by pharma companies→ detects seizure liability

• The architecture and anatomy of the hippocampus is well defined

• Recordings conducted from CA1 neurons (Involved in learning and memory; also a focus for seizures in the brain)

• Stimulation of afferent input (Schaffer collateral commisural pathway)

  • 1. Stimulus artefact

  • 2. Control population spike with a negative-going potential

  • 3. positive-going potential

• Application of convulsant drug → epileptic population spike

  • Initial part of trace resembles control population spike followed by many depolarisations

• Procedure is gradually being replaced by neuronal cultures

How are microelectrode arrays (MEAs) used in in vitro neurotoxicity assessment?

• Neuronal cultures (or co-culture) with glial cells are grown on microelectrode arrays (MEAs)

• Use stimulation and recording electrodes to measure activity

• Example:

  • Bicuculline & gabazine (GABAᴀ competitive antagonists) → increase in burst firing of glutamatergic neuron

  • PTZ (GABAᴀ non-competitive antagonist) → no effect

• This shows that systems are not yet fully optimised

• MEAs are emerging in vitro seizure assays that are beginning to replace rat hippocampal slices.

What is 24/7 home cage monitoring in group-housed rats?

• Video camera used to monitor behaviour in a cage

• RFID chip is implanted under skin of the ventral abdomen in the rat

• RFID reader present under cage tracks movement and allows 24/7 monitoring of an individual in a group-housed conditions

• Measures:

  • Activity levels

  • Circadian rhythms → rhythmic activity shows an increase at night/dark phase

  • Temperature changes → activity shows an increase at night/dark phase

• Findings show a short-lived increase in activity (run, eat, groom, sleep) during dark phase

• Used for in vivo behavioural and neurotoxicity assessment

• This system has been gradually adopted and adapted for different cage types

How is Motor Co-Ordination Assesed in In Vivo Neurotoxicity Assessments?

• Non-invasive and non-stressful test

• Incorporated into ongoing toxicity studies and conducted on the main study animals

• Beam walking:

  • Rats trained to walk across a beam in a brightly lit room

  • Walk towards the dark box

  • Monitor footfall as the rats walk across the 1m beam

• Gate analysis:

  • Rats trained to walk across a glass corridor

  • When rats stand on the glass corridor, the light is projected through the glass

  • When the animal makes contact, there is light scattering at the site of the paws → detected by a camera under the glass

  • Provides information on walking speed, sway around the midline and alterations in gait

• Accelerating rotarod

  • Animals are trained to walk forward on a spindle until they can no longer maintain their balance

  • If they fall off onto a switch which stops the clock

• These are simple forms of technology that are sensitive to detecting effects on the nervous system

What did the accelerating rotarod study show in repeat-dose toxicology in rats?

• A drug for T2DM caused hypoglycaemia-induced neuropathy and recovery in normal rats

• After 4 weeks of dosing, the rats had low glucose resting levels

• Decreased rotarod performance in hypoglycaemic rats

  • (Vehicle dosing performance is stable)

• Deficit preceded adverse clinical neurological signs during standard clinical observations

• Rotarod deficit correlated with histopathology of damaged peripheral (sensory) nerves

• Recovery occurred ~10 weeks after the 4-week dosing period stopped (hangover effect)

  • Long recovery period

• Demonstrates indirect neurotoxicity via hypoglycaemia

What are the two components of Sensory Neuropathy?

• Hyperalgesia: increased sensitivity to noxious stimuli e.g. putting hands in ice water

• Allodynia: Sensitivity to non-noxious stimuli

• These are both generally associated with sensory neuropathy (takes 24 hours to develop, e.g. paclitaxel)

What Nociception Tests are Used to Assess Sensory Neuropathy?

• Methods effective at detecting the effect on drugs causing hyperalgesia or allodynia

• Tail flick test: tail placed over the infrared heat source

  • When heat is detected as noxious, the tail is moved → stops the source and the clock

• Thermal planar test: A similar test done under the skin of the hind paw

  • An animal lifts its paw when it becomes noxious → stops the clock

• Paw pressure test: use of pressure as stimulus

  • A stylus is placed by the side of the hind paw which increase pressure until the animal withdraws the paw and removes the pressure

How are Electroencephalography (EEG) used in in vivo seizure detection and neurotoxicity assessment?

• Mouse, rat, or guinea pig implanted with an EEG telemetry device under anaesthesia

• After several days of recovery, recordings are made in conscious, freely-moving animals

• Convulsant agents that induce seizures cause characteristic spike and wave changes on the EEG

  • This is often accompanied by behavioural manifestations

How are Electroretinography (ERG) used to assess retinal dysfunction in vivo?

• ERG measures retinal cell function using a flashing stimulus and a contact lens electrode

• A-wave: photoreceptors’ activity (with possible input from retinal epithelial cells)

• B-wave: bipolar + Müller cell activity; Oscillatory potentials (OPs): amacrine cells

  • Muller cells: glial cells that span the length of the retina

• Example:

  • Low dose of drug → no effect

  • Intermediate and high dose → loss of B-wave

    • a-wave largely unaffected

• Indicates toxicity affecting bipolar/Müller cells

• Test conducted in complete darkness and used to identify the cell-type-specific retinal dysfunction induced by the drug

Give an Example of A Drug that Induced Retinal Dysfunction

• Monocarboxylate-1 inhibitor (AZD3965)

• Developed for cancer therapy → tumours are glycolytic and thus succeptible to lactate uptake inhibitors

• Loss of B-wave at higher doses

• Demonstrates retinal toxicity

How is Auditory Brainstem Response (ABR) used to assess auditory neurotoxicity in vivo?

• Scalp electrodes record auditory brainstem responses under anaesthesia

• Sound stimulus (standard tone or sudden brief noise) delivered via loudspeaker

• Measure threshold sound level (dB) evoking ABR at multiple frequencies (e.g. 4, 10, 20 kHz)

• Aims to detect the level of noise required for an effect to be observed

• Increase ABR threshold = hearing impairment

What Does Auditory Brainstem Response (ABR) reveal about Furosemide auditory toxicity in vivo?

• Furosemide , a standardised diuretic agent used (100 mg/kg, IV)

• Causes reversible hearing loss

• Shows an increased ABR threshold at all 3 frequencies (4, 10, 20 kHz)(**P<0.01)

What are the Emerging Soluble Biomarkers in In Vitro and In Vivo Neurotoxicity Assessments?

• Cell body

  • Ubiquitin Ci-terminal hydrolases-L1

  • miR-9

  • miR-385-5p

  • Neuron-specific enolase

• Dendrites

  • MAP-2

• Axons

  • microtubule-associated protein TAU

  • Spectrin breakdown product (SBDP-145)

  • Neurofilament light chain (NF-L)

• Astrocytes

  • Glial fibrillary acidic protein (GFAP)

  • S-100B

• Activated glia

  • Translocator Protein (TSPO)

• Myelin sheath

  • myelin basic protein

• With the emergence of better bioanalytical techniques, these biomarkers can be detected in CSF and plasma

How is the Soluble Biomarker NFL-1 Used to Assess Neurotoxicity in Vivo?

• Biomarker used to assess axonal neurotoxicity in rats

• Measured in blood

• Example (rat study):

  • Day 4 → little effect

  • Day 8 & 29 → increase in NF-L1 levels

• Demonstrates sensitivity of assay with detection of neuronal damage in blood

How is Neroimaging Used in In Vivo Neurotoxicity Assessments?

• Detection of neurochemical changes in vivo using magnetic resonance spectroscopy (MRS)

• Decrease in myoinositol specific to the anterior cingulate cortex in welders’ chronic exposure to Manganese → Change correlates with cognitive deficit

• Absent feature on the waveform in welders vs controls

• Offers an in vivo non-invasive method of assessing CNS damage

What Compounds has Neroimaging in rats been used for Neurtox Evaluations?

• MDMA (ecstacy)

• (+)-methamphetamine

• Anandamide

• Acrylamide

• 1,3-dinitrobenzene

• MK801

• Hydrazine

• Manganese

• 3-nitroproprionic acid

How is Histopathology Used in Post-Mortem Neurotoxicity Assessments?

• Used for routine assessment

• Several coronal sections are taken through the neural axis throughout the brain

• If neurotoxicity is anticipated, the number of sections taken increases

What are organophosphate acetylcholinesterase inhibitors and why are they significant?

• Potent, irreversible acetylcholinesterase inhibitors (bind to the esteric site and form covalent bonds)

• Developed as chemical warfare agents

• Classified as weapons of mass destruction by the United Nations

• All stockpiles were banned under the Chemical Weapons Convention (Multinational treaty) in 1993

• Have re-emerged in recent years

What is Acetylcholine?

• A neurotransmitter in the CNS

• Primary neurotransmitter in motor neurons, skeletal muscles and diaphragm (released at NMJ)

• Primary neurotransmitter at ganglia (synapses) of the ANS → executes unconscious function, e.g.

  • maintenance of smooth muscle tone, in the cardiovascular system, airways, bladder and GI tract, heart rate and exocrine glands

• Primary neuroeffector for the parasympathetic branch of the ANS

• Also has non-neurotransmitter roles elsewhere in the body

• Released by neurones; broken down by AChE

  • If AChE is inhibited → consequences are widespread and lethal

What are the Pathophysiological Consequences of Cholinesterase Poisoning?

• The sustained augmentation of cholinergic functions causes:

  • Nausea & vomiting

  • Dizziness

  • Salivation

  • Lacrimation

  • Pupillary constriction (miosis)

  • Bladder incontinence

  • Diarrhoea

  • Sweating

  • Impaired motor function; weakness; fatigue

  • Myoclonic spasms

  • Respiratory difficulties

  • Convulsions

  • Coma

• Death follows respiratory paralysis

• Seen following consumption of unwashed vegetables exposed to pesticides containing AChEis

What are Tabun, Sarin, Soman and Cyclosarin?

• Chemical warfare agents developed in Germany from 1936 (tabun) onwards (cyclosarin post-war in 1949).

• They are simple molecules

• Tabun, sarin and soman were stockpiled but never used by the German military.

• Producing or stockpiling agents banned by the Chemical Weapons Convention (1993)

• As of December 2015, most of the stockpiles had been destroyed.

How Have These Chemcial Warfare Agents (Tabun, Sarin, Soman and Cyclosarin) Been Used as Weapons?

• Following World War II the USA and the Soviet Union stockpiled these agents.

• In 1988: Iraq used sarin, cyclosarin and tabun in aerial attack on Kurdish civilians in Halabja, killing 5,000 people.

• In 1994, a religious sect released sarin into the atmosphere in central Matsumoto, killing 7 people and affecting ~600 others.

  • The following year, the same group released sarin on the Tokyo underground, killing 13 people.

• From 2013 onwards: Reports of use of sarin against civilians in the conflict in Syria.

  • Suspected to have been killed by sarin delivered by shells in Syria (2014)

What is Venomous Agent X (VX)?

• A chemical warfare agent developed in the UK in 1950s

  • Inadvertent creation from the pesticide programme at ICI

• More potent than sarin

• Readily absorbed via the skin

• Stockpiled by the USA, the Soviet Union, Syria and North Korea

• Stockpiles ordered for destruction under a multi-lateral treaty in 1993

• Used on 13th Feb 207 attack on Kim Jong-nam (NK leader kim jong-un half brother) → VX thrown on face in Kuala Lumpur International Airport; died with 20 minutes of attack

What is Novichok?

• A series of nerve agents developed by the Soviet Union and Russia between 1971 and 1993

• Up to 8× more potent than VX nerve agent

• Readily absorbed through the skin

• Notable incident of Uses:

  • March 2018 Salisbury poisoning of Sergei Skripal and daughter Yulia (Former GRU officer)

    • Large areas of the town required decontamination; 24 emergency service vehicles were buried in landfill sites

  • Death of Dawn Sturgess (Salisbury resident) → poisoning following spraying herself with perfume from a discarded bottle found by her boyfriend

  • 2020 poisoning of Alexei Navalny (Russian Opposition Politician) during an international flight

• Classified as a highly potent chemical warfare nerve agent