Botox Reconstitution Guide

Over the last two decades, Botox® has revolutionized medical aesthetics, in addition to its indications for overactive bladder, migraine, and muscle dystonia. Worry lines, laugh lines, crow’s feet, and frown and laugh lines can be safely and quickly diminished with Botox® injections to the face [1]. The dosages used for botulinum toxin injections for cosmetic purposes are far lower than those for other medical injectables. 

To ensure safe and successful outcomes, physicians aim to adjust the smallest effective dose to maintain precision, especially in medical aesthetics [2]. Many doctors have started using more sterile saline while reconstituting Botox®. This makes determining how many units to provide at each dose point more leisurely [3]. The use of Botox® can be for cosmetic treatments, as an acetylcholine release inhibitor, and as a neuromuscular blocking agent [4, 5].  

This article elaborates on the instructions for healthcare professionals to achieve accurate Botox®  reconstitution and dilution ratio and maintain efficient application. 

Key Takeaways

• Dosages: The dosages used for botulinum toxin injections for cosmetic purposes are far lower than those for other medical operations.  
• Products: Botox® vials are available in 50 or 100 powdered dose units. Subcutaneous injection requires reconstitution with saline. The results are generally obtained within a week.  
• Usage Timeframe: The product should be used within 24 hours of reconstitution, and the proportions should be colorless, transparent, and solids-free. 
• Side Effects: The risk of problems and adverse events is the same with this technique as it is with any injectable therapy. Cosmetic botulinum toxin injections have not been associated with any significant adverse effects. 

Overview of Therapeutic Applications

The fact that Botox® blocks the release of acetylcholine by sympathetic cholinergic neurons led to its evaluation for hyperhidrosis and excessive perspiration by medical professionals [6]. Furthermore, using injections to treat laryngeal dystonia sparked a further investigation into the innervation of other sphincters in the gastrointestinal system, leading physicians to focus on treating pelvic sphincters. 

Overactive bladder, neurogenic detrusor overactivity, achalasia, obesity, postoperative atrial fibrillation, and other autonomically mediated diseases were the subjects of further study [7]. 

Additionally, the cosmetic experience led to the discovery that studying Botox® (onabotulinumtoxinA) for depression could enhance mood by relaxing the facial muscles linked with unpleasant emotions. 

Formal development programs and post-marketing reports have demonstrated the safety profile of onabotulinumtoxinA for recognized uses. Although rare, mounting evidence shows systemic effects can appear clinically, especially at high dosages and in susceptible populations [8]. 

Botox® Reconstitution Ratios

Botox® vials are available in 50 or 100 powdered dose units. Subcutaneous injection requires reconstitution with saline. The results are generally obtained within a week. 

Areas of the face prone to fine muscle wrinkling and line formation are common injection sites for reconstituted Botox®. It has numerous uses, including reducing wrinkles around the nose and chin, although it is most commonly used on the area between the brows (glabellar lines) and the corners of the eyes (canthal lines) [9].  

It is standard practice to dilute Botox® with saline before injecting it, regardless of whether the goal is medicinal or aesthetic. In its original vial form, Botox® is a dry powder. The healthcare provider dilutes it by slowly adding the correct amount of saline to the drug vial using a syringe and spinning it gently to mix it.

The product should be used within 24 hours of reconstitution, and the proportions should be colorless, transparent, and solids-free [10].  

Diluent* added to 100 Unit Vial	Resulting dose units per 0.1ml	Diluent added to 200 Unit Vial	Resulting dose units per 0.1ml
1ml	10 units	1ml	20 units
2ml	5 units	2ml	10 units
4ml	2.5 units	4ml	5 units
8ml	1.25 units	8ml	2.5 units
10ml	1 unit	10ml	2 units

*Preservative-free 0.9% Sodium Chloride Injection, USP Only  

It is common practice to administer a 20-unit dosage in four little injections. Injecting 0.1 milliliters of reconstituted saline per site instead of 0.04 milliliters with a 1.0-milliliter dilution is recommended for a 100-unit saline vial.

With a capacity of only 0.1 milliliters, the physician has a more straightforward reading measurement and precisely dosing each injection across the necessary area. Additionally, the tiny “dead space” in the hubs of all hypodermic needles renders less active Botox® dose. This issue becomes even more noticeable in the minimal amounts utilized in cosmetics [11].  

Using smaller-volume syringes can further simplify dosage readings. Because the dose marks on lower amounts are further apart, many injectors choose 3ml or 5ml syringes. This allows for more precise control with the little plunger movements and makes reading more manageable for the human eye.

Some doctors are worried that botulinum will spread further in the body with bigger-volume injections. Even though the danger of migration increases with increasing injection volume, many practitioners believe that the advantages of better dosage control and placement more than justify the slight increase to 2.5 ml of dilution [11, 12].  

Some patients and doctors have expressed concern about the safety of using more diluted Botox®. The immense volume is more straightforward to manage precisely in the syringe, improving safety even if the total Botox® dose remains unchanged. Using this method, overdosing at specific injection sites can be more easily prevented [11, 12].  

The recommended dosage of Botox® Cosmetic administration ranges between 20 and 40  Units, not exceeding a total dose of 400 Units in a 3-month interval [4]. The dose of  Botox®, when used as an acetylcholine release inhibitor and a neuromuscular blocking agent, should not exceed 400 Units in adults (Zable 2) and 10 Units/kg or 340 Units in children.

Botox® Reconstitution Ratios for Pediatric Patients

Body Weight(kg)	Volume of reconstituted BOTOX and Diluent (mL) to draw into the dosing syringe to achieve a final volume of 10 mL		Final dose of BOTOX in dosing
	BOTOX (mL)	Diluent (mL)
12 to less than 14	3.6	6.4	72 Units
14 to less than 16	4.2	5.8	84 Units
16 to less than 18	4.8	5.2	96 Units
18 to less than 20	5.4	4.6	108 Units
20 to less than 22	6	4	120 Units
22 to less than 24	6.6	3.4	132 Units
24 to less than 26	7.2	2.8	144 Units
26 to less than 28	7.8	2.2	156 Units
28 to less than 30	8.4	1.6	168 Units
30 to less than 32	9	1	180 Units
32 to less than 34	9.6	0.4	192 Units

Dilution Process

The number of units per milliliter varies depending on the amount of dilution. The amount of dilution is decided according to the area to be applied and the concentration of toxin planned to be used. Preservative-free serum should be used for dilution purposes. 

Since the amount of pain felt during injection is significantly higher in dilutions made with sterile distilled water, this should be avoided. During dilution, the serum should be injected slowly into the vial, and foaming or bubbling that may cause denaturation of the toxin should not be allowed. For this purpose, it is preferable to perform the dilution process by opening the vial cap and eliminating the negative pressure effect [13]. 

Storage of Diluted Botulinum Toxins

Botulinum toxin should be stored frozen until prepared, but not frozen again after dilution. The diluted toxin can be stored at +4 degrees until used. Using diluted toxin as soon as possible (within the first 4 hours) is ideal [14]. 

Although there is information in the literature that diluted Botox can be used for 1 month in the refrigerator without any significant decrease in its effect, ultrastructural studies conducted in recent years have shown that the effectiveness of the stored toxin decreases [15].  

Efficacy and Safety

In most cases, botulinum toxin has been well-tolerated and safe for therapeutic usage.  Minor, fleeting, and eventually gone are the characteristics of adverse consequences. The risk of problems and adverse events is the same with this technique as it is with any injectable therapy. 

Cosmetic botulinum toxin injections have not been associated with any significant adverse effects. Because the application method determines the difficulty level, proper training and mastery of the technique are crucial [16].  

The drug’s diffusion to non-target muscles is another possible mechanism by which these effects manifest; the specific product and treatment area must be considered. As a result,  the site of the clinical impact may change, and the severity of the side effects may rise. 

Because of its complex nature, botulinum toxin can migrate unintentionally, affecting neighboring muscles and causing undesirable side effects. Area of application, dilution volume, product specifics, dosage, and administration method are some elements that affect the botulinum toxin migration profile [16]. 

Potential Side Effects

Patients undergoing Botox® injections may experience some discomfort and fear that the increased fluid content can lead to ecchymosis (purple coloring), bruising, or worse pain.  Needle design and injector technique are the leading causes of pain, discoloration, and bruising. Patients report less pain and fewer adverse effects after the introduction of new ultra-fine “invisible” needles [14].  

Conclusion

Botox treatment works by temporarily relaxing the muscles to which it is applied. The patient’s muscle mass, wrinkle degree, anatomical structure, and skin thickness determine the amount of medication used and the application areas.

Dilution is performed with 0.9% physiological serum without preservatives. The patient’s expectations should coincide with the physician’s application skills and technique. Accurate dilution, dose adjustment, and compliance with basic instructions prior to administration are two of the key factors of procedure success. 

FAQ

What is the repetition of Botox treatment?  

Botox applications are repeated when the effect wears off, which is, on average,  every 4-6 months. This period varies and may be extended depending on the person’s muscle structure and the number of Botox sessions they have had before. As Botox sessions increase, the muscle weakens, facial expressions decrease, and a lower dose can achieve the same effect.  

Can you combine Botox with other applications?  

Combined applications can be made with botulinum toxin. Filling can be done in the same session with thread-suspension. It is not recommended to have it in the same session with PRP CGF STEM cell and to mix it. It can only be mixed with mesotherapy products as a product.  

Can you use Botox for every patient?  

It is not applied to pregnant women, breastfeeding women, people with severe systemic diseases such as autoimmune disease or cancer, people with toxin allergies or anaphylactic reactions, or people with active skin diseases, systemic infections, and local infections in the application area.  

What are the risks of Botox treatment?  

After the application, bruising may sometimes occur at the injection sites, which will disappear within a few days. Botox application does not cause tumors or cancer. The application itself does not cause addiction or dependency. 

References 

• Hall C. Optimizing Facial Aesthetics: Sequential Application of Botulinum Toxin 4 and  Dermal Fillers for Enhanced Results. Plast Aesthet Nurs (Phila). 2024,44(1):70-71.  doi:10.1097/PSN.0000000000000546  
• Bavikatte G, Esquenazi A, Dimyan MA, et al. Safety and Real-World Dosing of  Onabotulinumtoxind for the Treatment of Adult Spasticity: Post Hoc Analysis of the  Adult Spasticity International ~ Registry Study. Am J Phys Med Rehabil.  2024;103(7):580-587. doi:10.1097/PHM.0000000000002410  
• Wang X, Zou ¥, Yuan M, Huang H, Han X, Gong X. Dose and injection site of botulinum  toxin type A for gummy smile management: A systematic review and bibliometric  analysis. Toxicon. 2024;249:108058. doi:10.1016/j.toxicon.2024.108058  
• Summary of Product Characteristics BOTOX® Cosmetic (onabotulinumtoxinA) is used  for injection and intramuscular use. Allergan, Revised: 8/2023  
• Summary of Product Characteristics BOTOX® Cosmetic (onabotulinumtoxind) for  injection, for intramuscular use, intradetrusor, or intradermal use. Allergan, Revised:  Revised: 10/2024  
• Pozo-Rosich P, Alpuente A, Silberstein SD, Burstein R. Insights from 25years of  onabotulinumtoxind in migraine – mechanisms and management. Nat Rev Neurol.  2024;20(9):555-568. doi:10.1038/s41582-024-01002-5  
• Brin MEF, Blitzer A. The pluripotential evolution and journey of Botox  (onabotulinumtoxind). Medicine (Baltimore). 2023;102(S1):e32373.  doi:10.1097/MD.0000000000032373  
• Borba A, Matayoshi S, Rodrigues M. Avoiding Complications on the Upper Face  Treatment With Botulinum Toxin: A Practical Guide. Aesthetic Plast Surg.  2022;46(1):385-394. doi:10.1007/s00266-021-02483-1 
• Brin MF, Burstein R. Botox (onabotulinumtoxind) mechanism of action. Medicine  (Baltimore). 2023,102(S1):e32372. doi:10.1097/MD.0000000000032372  
• Understanding ~ Botox® Reconstitution — [With ~ Chart] htips://www.air-tite 10. shop.com/Articles/botox-reconstitution-with-chart?srsltid=AfimBOoo2ey3djSeviwDaDF4FMojOWahwZUFkmS ViB5SVGSFMFF  OwOKE, [Accessed at 09/12/2024]  
• Botulinum Neurotoxin Type A According to Storage Temperatures. Dermatol Surg.  2020;46(12):1657-1660. doi:10.1097/DSS.0000000000002557  
• Elmas C, Avhan S, Tuncer S, et al. Effect of fresh and stored botulinum toxin a on muscle  and nerve ultrastructure: an electron microscopic study. Ann Plast Surg.  2007;59(3):316-322. doi:10.1097/SAP.0b013e31802e0912
• Brin MF, De Boulle K, Liew S, et al. Safety and tolerability of onabotulinumtoxinA in  the treatment of upper facial lines from global registration studies in 5298 participants:  A meta-analysis. JAAD ~ Int. 2023;14:4-18. Published ~ 2023 Sep 9.  doi:10.1016/j.jdin.2023.07.021 
• Boudreau G, Finkelstein I, Graboski C, et al. Onabotulinumtoxin4 Improves Quality of  Life in Chronic Migraine: The PREDICT Study – CORRIGENDUM. Can J Neurol Sci.  2024;51(1):149. doi:10.1017/cjn.2023.317