Provocholine (Methacholine Chloride)
Diagnostic agent for bronchial provocation testing to assess airway hyperresponsiveness
Provocholine is a prescription diagnostic medicine containing methacholine chloride. It is used exclusively for bronchial provocation testing (methacholine challenge test) to help diagnose non-specific bronchial hyperresponsiveness in patients suspected of having asthma. The test must be performed under the supervision of a specialist physician with emergency resuscitation equipment immediately available.
Quick Facts
Key Takeaways
- Provocholine is used exclusively for diagnostic purposes – it is not a treatment for asthma or any other condition.
- The methacholine challenge test detects bronchial hyperresponsiveness by measuring the dose that causes a 20% fall in FEV1 (PD20 or PC20).
- Testing must be supervised by a specialist physician with emergency resuscitation equipment immediately available.
- Several medications including beta-agonists, antimuscarinics, and theophylline must be withheld before the test to avoid false-negative results.
- Provocholine is contraindicated during pregnancy and should be used with caution in patients with epilepsy, cardiovascular disease, or peptic ulcers.
What Is Provocholine and What Is It Used For?
Provocholine (methacholine chloride) is a cholinergic agonist used exclusively in diagnostic medicine. It belongs to the ATC category V04C (other diagnostic agents) and is classified as a diagnostic tool rather than a therapeutic drug. Its sole clinical purpose is to perform bronchial provocation testing, also known as a methacholine challenge test, which is one of the most widely used methods for assessing airway hyperresponsiveness.
The methacholine challenge test is a well-established diagnostic procedure recommended by the European Respiratory Society (ERS) and the American Thoracic Society (ATS) for the evaluation of patients with suspected asthma. The test is particularly useful when patients present with symptoms suggestive of asthma – such as intermittent cough, wheezing, or chest tightness – but have normal baseline spirometry results. In these cases, a negative methacholine challenge test has a high negative predictive value, meaning it can effectively rule out asthma as a diagnosis.
Methacholine works by stimulating muscarinic receptors on the smooth muscle of the airways. When inhaled in increasing concentrations, it causes dose-dependent bronchoconstriction. Patients with bronchial hyperresponsiveness, such as those with asthma, are significantly more sensitive to this bronchoconstricting effect compared to healthy individuals. By measuring the dose or concentration at which a patient's FEV1 (forced expiratory volume in one second) decreases by 20% from baseline, clinicians can quantify the degree of airway hyperresponsiveness.
Provocholine is approved for use in adults and children aged 5 years and older who are capable of performing reproducible spirometry. The patients being tested do not need to have clinically apparent asthma, but they should have a clinical history suggestive of the condition alongside normal spirometry results. The test result is expressed as either PD20 (provocation dose causing 20% FEV1 fall) or PC20 (provocation concentration causing 20% FEV1 fall), which provides a standardised measure of airway reactivity.
Provocholine is intended for diagnostic use only. It is not a treatment for asthma or any other respiratory condition. The bronchoconstriction caused by the test is temporary and reversible with a short-acting beta-agonist bronchodilator administered after the procedure.
What Should You Know Before Using Provocholine?
Before undergoing a bronchial provocation test with Provocholine, it is essential that your physician obtains a comprehensive clinical respiratory history. This is important because false-positive test results can occur in a number of non-asthmatic conditions. Conditions such as recent influenza, upper respiratory tract infections, recent immunisations, and chronic lung diseases (including cystic fibrosis, sarcoidosis, tuberculosis, and chronic obstructive pulmonary disease) may all produce positive results on methacholine challenge testing without the patient actually having asthma.
Additionally, patients with allergic rhinitis without asthma, current smokers, and those exposed to significant air pollution may also show positive results. For these reasons, the clinical context must always be considered alongside the test result when making a diagnostic conclusion. A positive methacholine challenge test alone does not confirm asthma; it confirms bronchial hyperresponsiveness, which can have multiple causes.
Contraindications
Provocholine must not be used during pregnancy. No animal studies have been conducted to evaluate reproductive toxicity of methacholine chloride, and it is unknown whether the drug can cause foetal harm. Furthermore, the bronchoconstriction induced by the test could potentially compromise oxygen supply to both the mother and the developing foetus, which could be harmful.
It is unknown whether methacholine chloride is excreted in breast milk. Caution should therefore be exercised when administering Provocholine to breastfeeding women. The physician should carefully weigh the diagnostic benefit against any potential risk.
Warnings and Precautions
The administration of Provocholine must only be performed under the supervision of a specialist physician who is thoroughly familiar with all aspects of methacholine inhalation challenge test technique, including all contraindications, warnings, precautions, and the management of respiratory distress. The supervising physician must be contactable during the test and immediately available if needed. If the physician is conducting the test personally, another person must be available to assist if required. The patient must never be left unattended during the procedure.
Severe bronchoconstriction and significant reduction of respiratory function can occur as a result of Provocholine administration. Patients with airway hyperresponsiveness may experience bronchoconstriction at doses as low as 0.031 mg/ml. Emergency resuscitation equipment and acute medications must be immediately available to treat respiratory distress. If severe bronchoconstriction occurs, it must be promptly treated with inhaled short-acting beta-agonist bronchodilator.
The test requires accurate baseline spirometry. If baseline spirometry is not performed or measured accurately, and the initial FEV1 is underestimated, subsequent decreases after inhalation of Provocholine solutions may not be detected, resulting in administration of doses that are too high and excessive bronchoconstriction. Similarly, unacceptable test manoeuvres can produce false-positive results.
Special caution is required when administering Provocholine to patients with any of the following conditions:
- Epilepsy – cholinergic stimulation may lower seizure threshold
- Cardiovascular disease – vagal stimulation may affect cardiac function
- Vagotonia – enhanced parasympathetic response
- Peptic ulcer disease – cholinergic agents may increase gastric acid secretion
- Thyroid disease – potential for exacerbated autonomic effects
- Urinary tract obstruction – cholinergic stimulation may worsen obstruction
In these patients, Provocholine should only be administered if the physician determines that the benefit-risk ratio is favourable. Patients with asthma are considerably more sensitive to methacholine-induced bronchoconstriction than healthy individuals, and particular care is required for patients with unstable asthma.
Laboratory personnel with asthma or allergies should exercise particular caution and take necessary precautions when handling the material or when performing the test on patients. The powder should not be inhaled, and personnel should avoid direct handling of the drug.
Pregnancy and Breastfeeding
Provocholine must not be used during pregnancy. No animal reproduction studies have been conducted with methacholine chloride, and it is unknown whether the drug can cause foetal harm when administered to pregnant women. Inadequate oxygen supply during pregnancy due to bronchoconstriction could be harmful to the foetus.
It is unknown whether methacholine chloride is excreted in human breast milk. A decision must be made whether to discontinue breastfeeding or to postpone the diagnostic test, taking into account the clinical need for the diagnostic information.
The effects of methacholine chloride on fertility are unknown. No studies have been conducted to evaluate this aspect.
Use in Children
Provocholine is approved for use in children aged 5 years and older who are capable of performing reproducible spirometry. However, clinicians should be aware that children may also show positive results due to non-asthmatic increased airway responsiveness. This means that a positive result in a child does not automatically confirm asthma. It is important to consider other possible respiratory conditions in the paediatric population when interpreting test results.
The same safety precautions apply to paediatric patients as to adults, including the requirement for specialist supervision, available emergency equipment, and adequate baseline spirometry.
How Does Provocholine Interact with Other Drugs?
A number of medications used in the treatment of asthma can suppress airway hyperresponsiveness to methacholine, potentially producing false-negative results. These medications must be discontinued before the provocation test, and the required withholding period depends on the duration of action of each specific drug. The following table summarises the minimum withholding times recommended for commonly used respiratory medications.
Medications That Reduce Airway Hyperresponsiveness
| Medication | Example | Minimum Withholding Period |
|---|---|---|
| Short-acting beta-agonists (conventional inhaled doses) | Salbutamol 200 µg | 6 hours |
| Long-acting beta-agonists | Salmeterol | 36 hours |
| Ultra-long-acting beta-agonists | Indacaterol, vilanterol, olodaterol | 48 hours |
| Short-acting antimuscarinics | Ipratropium (Atrovent) 40 µg | 12 hours |
| Long-acting antimuscarinics | Tiotropium, umeclidinium | ≥ 168 hours (7 days) |
| Oral theophylline | Theophylline preparations | 12–48 hours |
Medications That Do Not Require Withholding
Cromones, inhaled corticosteroids, and leukotriene modifiers have little or no effect in single doses and do not need to be withheld before the test, unless the intention is to assess the presence of an anti-inflammatory effect. After regular use, the duration of their effect on airway responsiveness is uncertain, but a washout period of 4–8 weeks is considered reasonable if their anti-inflammatory impact needs to be assessed.
Dietary Considerations
Normal dietary intake of caffeine and caffeine-related products (such as chocolate, coffee, and tea) has no clinically significant effect on methacholine challenge test results and does not need to be restricted. However, tobacco smoking should be avoided for at least one hour before the test, as acute smoking can alter airway responsiveness.
The methacholine challenge test should only be considered for patients who take asthma medications regularly if the accuracy of the diagnosis is questionable. The test should be performed after supervised discontinuation of asthma medications and provided that normal spirometry is demonstrated. Clinicians should also consider the possibility of rebound airway hyperresponsiveness following withdrawal of asthma medications when interpreting test results.
What Is the Correct Dosage of Provocholine?
Provocholine is supplied as a 100 mg powder in a 20 ml vial. It must be reconstituted and diluted with 0.9% sodium chloride for injection in empty sterile borosilicate glass vials (Type I). The vial should be shaken after adding the sodium chloride solution until a clear solution is obtained. All dilutions must be filtered through a sterile hydrophilic bacterial retention filter with a pore size of 0.22 µm when transferring the solution from each vial (minimum 2 ml) to the nebuliser.
Adults and Children (5 Years and Older)
Provocholine is administered by inhalation only. Before beginning a provocation test, baseline spirometry must be performed. The patient must have a baseline FEV1 of at least 60% of the predicted value (in both adults and children) and at least 1.5 litres (in adults). The post-diluent FEV1 must also be measured before starting Provocholine administration.
Two dosing protocols are available: doubling doses (more precise PD20 values, recommended when monitoring changes in airway reactivity after treatment) and quadrupling doses (recommended for routine clinical testing, as it is faster with fewer inhalation steps).
Preparation of Serial Dilutions – Doubling Concentrations
| Take | Add 0.9% NaCl | Concentration | Dose |
|---|---|---|---|
| 100 mg Provocholine | 6.25 ml | 16 mg/ml (A) | 380 µg |
| 3 ml dilution A | 3 ml | 8 mg/ml (B) | 190 µg |
| 3 ml dilution B | 3 ml | 4 mg/ml (C) | 95 µg |
| 3 ml dilution C | 3 ml | 2 mg/ml (D) | 47.5 µg |
| 3 ml dilution D | 3 ml | 1 mg/ml (E) | 23.75 µg |
| 3 ml dilution E | 3 ml | 0.5 mg/ml (F) | 11.875 µg |
| 3 ml dilution G | 3 ml | 0.125 mg/ml (H) | 2.969 µg |
| 3 ml dilution H | 3 ml | 0.0625 mg/ml (I) | 1.484 µg |
| 3 ml dilution I | 3 ml | 0.0312 mg/ml (J) | 0.742 µg |
Preparation of Serial Dilutions – Quadrupling Concentrations
| Take | Add 0.9% NaCl | Concentration | Dose |
|---|---|---|---|
| 100 mg Provocholine | 6.25 ml | 16 mg/ml (A) | 380 µg |
| 3 ml dilution A | 9 ml | 4 mg/ml (B) | 95 µg |
| 3 ml dilution B | 9 ml | 1 mg/ml (C) | 23.75 µg |
| 3 ml dilution C | 9 ml | 0.25 mg/ml (D) | 5.938 µg |
| 3 ml dilution D | 9 ml | 0.0625 mg/ml (E) | 1.484 µg |
The doses listed correspond to the dose delivered from an English Wright nebuliser during two minutes of nebulisation, or the Hudson RCI MicroMist small-volume nebuliser during one minute of nebulisation, using dry compressed air with a pressure regulator set at 3.4 bar to produce output within 10% of 0.13 ml/min. Other suitable nebulisers may be used provided their output and particle size characteristics have been characterised. The acceptable mass median aerodynamic diameter (MMAD) is 1.0–3.6 µm.
Test Procedure (Tidal Breathing Method)
The tidal breathing method is the standard administration approach. The procedure is performed as follows:
- Draw up 2–3 ml of diluent (0.9% sodium chloride) and transfer it to the nebuliser. Connect the nebuliser and tubing to the compressed air source.
- Inform the patient that the aerosols may cause mild cough, chest tightness, or breathlessness. Instruct the patient to remove the face mask or mouthpiece and stop inhaling immediately if symptoms become uncomfortable.
- Have the patient breathe the diluent aerosol calmly (tidal breathing) for one minute while wearing the face mask loosely over the nose and mouth, or using a mouthpiece with a nose clip.
- After one minute, stop the nebuliser and measure FEV1 at 30 and 90 seconds after inhalation. If FEV1 after diluent decreases by 20% or more from average baseline FEV1, do not proceed with further inhalations.
- Begin administering Provocholine starting with the lowest concentration. For quadrupling dosing, start at 1.484 µg / 0.0625 mg/ml; for doubling dosing, start at 0.742 µg / 0.0312 mg/ml.
- Administer subsequent doses at 5-minute intervals, following the doubling or quadrupling dose increments.
- Continue until FEV1 has decreased by 20% or more from post-diluent FEV1, or the highest dose has been administered.
- After completing the test, administer two doses of a short-acting beta-agonist. Wait 10 minutes and measure FEV1 and vital capacity (VC). The patient must not leave the laboratory until FEV1 has returned to within 90% of baseline.
The ERS technical standard for bronchial challenge testing states that other suitable nebulisers may be used provided their output rate and particle size distribution have been characterised. When switching nebulisers with different characteristics, results should be reported as dose (PD20) rather than concentration (PC20) to improve test standardisation.
Interpretation of Results
The methacholine challenge test is considered positive if FEV1 decreases by 20% or more from the post-diluent FEV1 at any dose during the test. The result is quantified as PD20 (the provocation dose causing 20% FEV1 fall) or PC20 (the provocation concentration causing 20% FEV1 fall), calculated by log-linear interpolation between the last two data points.
A negative (normal) test is defined as a decrease in FEV1 of less than 20% after all doses have been administered. According to the ERS technical standard, a negative result is also defined as PD20 > 380 micrograms or PC20 > 16 mg/ml.
Overdose
Provocholine is administered by inhalation only. If accidentally administered orally or by injection, overdose with methacholine chloride can cause syncope with cardiac arrest and loss of consciousness. Severe toxic reactions should be treated with 0.5–1 mg atropine sulphate administered intramuscularly or intravenously.
What Are the Side Effects of Provocholine?
Like all medicines, Provocholine can cause side effects, although not everybody gets them. Side effects reported during clinical use of Provocholine were associated with 153 inhaled provocation tests with methacholine chloride. Most side effects are expected pharmacological responses related to the cholinergic mechanism of action and the resulting bronchoconstriction. They are generally transient and resolve after administration of a short-acting beta-agonist bronchodilator or spontaneously within 30–45 minutes.
The following side effects have been reported. Because the exact frequency cannot be calculated from available data, all are classified as “not known frequency”:
Respiratory, Thoracic and Mediastinal Disorders
Frequency not known (cannot be estimated from available data)
- Bronchoconstriction
- Bronchospasm
- Chest tightness
- Cough
- Wheezing
- Throat irritation
Nervous System Disorders
Frequency not known (cannot be estimated from available data)
- Headache
- Dizziness
Skin and Subcutaneous Tissue Disorders
Frequency not known (cannot be estimated from available data)
- Pruritus (itching)
The bronchoconstriction that occurs during methacholine challenge testing is the expected and desired pharmacological response – it is the basis of the diagnostic test. In most patients, lung function returns to normal within 5 minutes after administration of a short-acting bronchodilator, or within 30–45 minutes without a bronchodilator. Patients with significant airway hyperresponsiveness may experience bronchoconstriction at very low doses (as low as 0.031 mg/ml), which is why the test must always be conducted with appropriate medical supervision and emergency equipment readily available.
Although Provocholine is administered in a supervised clinical setting, inform the attending healthcare professional immediately if you experience severe breathing difficulty, persistent chest tightness, or any symptoms that do not improve promptly after administration of a bronchodilator. Emergency resuscitation equipment is always available during testing.
Healthcare professionals are encouraged to report any suspected adverse reactions to their national adverse drug reaction reporting system. Reporting helps in the continuous monitoring of the benefit-risk balance of the medicine.
How Should You Store Provocholine?
Provocholine powder in unopened vials does not require any special storage conditions. The vials should be stored in their original packaging until ready for use.
Once the powder has been reconstituted with 0.9% sodium chloride and serial dilutions have been prepared, the resulting solutions must be used immediately. All leftover reconstituted and diluted solutions should be discarded after the provocation test is completed. Do not store or reuse reconstituted solutions.
Keep all medicines out of the sight and reach of children. Do not use Provocholine after the expiry date stated on the vial label and carton. The expiry date refers to the last day of that month.
What Does Provocholine Contain?
Provocholine has a simple formulation consisting of a single active ingredient:
- Active ingredient: Each 20 ml vial contains 100 mg methacholine chloride
- Appearance: White powder for reconstitution into a nebuliser solution
- Diluent (not included): 0.9% sodium chloride for injection is used for reconstitution and serial dilutions
The product contains no other excipients. The reconstituted solution should be clear. The solution must be filtered through a 0.22 µm sterile hydrophilic bacterial retention filter before transfer to the nebuliser.
Manufacturer Information
Provocholine is manufactured by MetaPharm Inc., 81 Sinclair Blvd., Brantford, Ontario N3S 7X6, Canada. The marketing authorisation holder is ACIC Europe Limited, Leontiou 163, Clerimos Building, 2nd Floor, 3022 Limassol, Cyprus.
Does Provocholine Affect Driving or Operating Machinery?
Provocholine has no or negligible direct effect on the ability to drive and use machines. However, since the methacholine challenge test can cause temporary bronchoconstriction and potential symptoms such as cough, chest tightness, or breathlessness, patients should ensure that their lung function has returned to within 90% of their baseline FEV1 value before leaving the testing facility. In practice, most patients recover full lung function within 5 minutes after receiving a beta-agonist bronchodilator at the end of the test.
Frequently Asked Questions About Provocholine
A methacholine challenge test (also called a bronchial provocation test) is used to diagnose non-specific bronchial hyperresponsiveness, which is a hallmark feature of asthma. It is particularly useful for patients who have symptoms suggestive of asthma (such as intermittent cough, wheeze, or chest tightness) but show normal results on standard lung function tests (spirometry). The test involves inhaling increasing doses of methacholine while lung function is measured. A positive result (20% or greater drop in FEV1) confirms airway hyperresponsiveness, while a negative result has a high negative predictive value for ruling out asthma.
A typical methacholine challenge test takes approximately 30–60 minutes to complete, depending on whether doubling or quadrupling dose increments are used. Quadrupling doses involve fewer steps and are faster (approximately 30 minutes), while doubling doses provide more precise PD20 values but take longer (approximately 45–60 minutes). After the test, patients must remain in the laboratory until their FEV1 has returned to within 90% of baseline, which usually takes 5–10 minutes after receiving a bronchodilator.
A positive methacholine challenge test means that your airways showed increased sensitivity (hyperresponsiveness) to the methacholine, with your FEV1 falling by 20% or more from baseline. This is consistent with, but does not definitively prove, a diagnosis of asthma. False-positive results can occur in patients with allergic rhinitis, recent respiratory infections, chronic lung diseases, smokers, and those exposed to air pollution. Your physician will interpret the result in the context of your full clinical history and other test results.
The test is not painful. You breathe normally through a face mask or mouthpiece while inhaling the nebulised solution. Some patients may experience mild cough, a sensation of chest tightness, or shortness of breath during the test – these are expected responses and indicate that the airways are reacting to the methacholine. If symptoms become uncomfortable, you can remove the face mask or mouthpiece and stop inhaling immediately. After the test, a bronchodilator is given to quickly relieve any symptoms, and most patients feel completely normal within 5 minutes.
Many asthma medications need to be withheld before the test because they can suppress airway hyperresponsiveness and lead to false-negative results. Short-acting bronchodilators (e.g., salbutamol) must be withheld for at least 6 hours, long-acting bronchodilators (e.g., salmeterol) for at least 36 hours, and long-acting antimuscarinics (e.g., tiotropium) for at least 7 days. Your physician will provide specific instructions about which medications to stop and when. Never discontinue medications without your physician’s guidance, as abruptly stopping asthma treatment can be dangerous.
PD20 and PC20 are the two standard measures used to quantify airway responsiveness during a methacholine challenge test. PD20 is the provocation dose (in micrograms) of methacholine that causes a 20% fall in FEV1, while PC20 is the provocation concentration (in mg/ml) that produces the same response. Both are calculated by log-linear interpolation between the last two data points during the test. According to the ERS technical standard, a negative result is defined as PD20 > 380 micrograms or PC20 > 16 mg/ml. Lower values indicate greater airway hyperresponsiveness.
References
This article is based on the following evidence-based sources:
- Coates AL, Wanger J, Cockcroft DW, et al. ERS technical standard on bronchial challenge testing: general considerations and performance of methacholine challenge tests. European Respiratory Journal. 2017;49(5):1601526. DOI: 10.1183/13993003.01526-2016
- Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing – 1999. American Journal of Respiratory and Critical Care Medicine. 2000;161(1):309–329. DOI: 10.1164/ajrccm.161.1.ats11-99
- Global Initiative for Asthma (GINA). Global Strategy for Asthma Management and Prevention. Updated 2024. Available at: ginasthma.org
- National Institute for Health and Care Excellence (NICE). Asthma: diagnosis, monitoring and chronic asthma management. NICE guideline [NG80]. Updated 2021.
- World Health Organization (WHO). WHO Model List of Essential Medicines. 23rd list, 2023.
- Provocholine Summary of Product Characteristics (SmPC). ACIC Europe Limited. Last updated 2020.
- Sterk PJ, Fabbri LM, Quanjer PH, et al. Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. European Respiratory Journal. 1993;6(Suppl 16):53–83.
Editorial Team
This article has been written and reviewed by the iMedic Medical Editorial Team, consisting of licensed specialist physicians in pulmonology, respiratory medicine, and clinical pharmacology.
Written by iMedic's medical content team with expertise in respiratory medicine and clinical pharmacology. All content follows the GRADE evidence framework and adheres to international guidelines from the ERS, ATS, and GINA.
Reviewed by the iMedic Medical Review Board – an independent panel of board-certified physicians who verify accuracy, completeness, and adherence to current evidence-based guidelines.
Evidence Standard: All medical claims in this article are supported by Level 1A evidence (systematic reviews and meta-analyses of randomised controlled trials) or current clinical guidelines from internationally recognised medical organisations. No commercial funding or pharmaceutical industry sponsorship has influenced this content.