Forehead markings used to measure frontalis muscle contraction. A, Subject showing markings made in the midpupillary line at 15 and 35 mm above the superior margin of the brow (ie, 20 mm apart). B, With maximum forehead contraction, the distance (x) between the 2 markings was measured, and movement was calculated in millimeters using the equation 20 − x.
Representative grid-like pattern used for control and experimental injections of botulinum toxin type A (Botox; Allergan Inc, Irvine, California).
Typical subject photographed prior to injection of botulinum toxin type A (Botox; Allergan Inc, Irvine, California) (A) and at each follow-up interval after injection: 2 weeks (B), 1 month (C), 2 months (D), 3 months (E), and 4 months (F). For each panel of photographs, comparisons are seen at rest (left) and with maximum forehead contraction (right).
Graphic comparison of average movement of the forehead sides injected with fresh vs 2-week refrigerated botulinum toxin type A (Botox; Allergan Inc, Irvine, California).
Graphic comparison of average movement of the forehead sides injected with fresh vs 2-week frozen botulinum toxin type A (Botox; Allergan Inc, Irvine, California).
Yang GC, Chiu RJ, Gillman GS. Questioning the Need to Use Botox Within 4 Hours of ReconstitutionA Study of Fresh vs 2-Week-Old Botox. Arch Facial Plast Surg. 2008;10(4):273-279. doi:10.1001/archfaci.10.4.273
Copyright 2008 American Medical Association. All Rights Reserved. Applicable FARS/DFARS Restrictions Apply to Government Use.2008
To determine whether injection with botulinum toxin type A (Botox; Allergan Inc, Irvine, California) reconstituted with preservative-free saline (0.9% isotonic sodium chloride) after 2-week cold storage in a refrigerator (4°C) or freezer (−20°C) is less efficacious than injection with freshly reconstituted Botox.
We conducted a prospective, double-blind, randomized controlled trial at an academic facial plastic surgery practice with 40 volunteers for treatment of horizontal forehead rhytids, each acting as his or her own control. In a blinded fashion each subject received freshly reconstituted Botox (control) on one side of the forehead (frontalis muscle) and 2-week-old reconstituted Botox (experimental) stored at 4°C (refrigerated) or stored at −20°C (frozen) on the other side. The right and left forehead movement was measured and photographed at rest and during maximum contraction of the frontalis muscle prior to Botox administration and on follow-up days 14, 30, 60, 90, and 120 after injection. Each participant also completed a questionnaire regarding right and left forehead movement prior to injection and at each follow-up visit.
No significant difference was noted for any subject in the timing of onset or duration of action or the measurable reduction of forehead movement between the fresh and 2-week-old refrigerated Botox or between the fresh and 2-week-old frozen Botox. The subjects had a gradual return of muscle function over the 4-month follow-up period.
No measurable difference was found in the potency or duration of efficacy of Botox in the treatment of forehead rhytids after 2 weeks of refrigeration or freezing compared with fresh reconstituted Botox. When Botox, fresh or stored, is given at an adequate dose to cause full paralysis of the desired muscle, the duration of the muscle paralysis is dependent on the physiologic rate for the motor nerve to reestablish neural transmission.
Botulinum toxin is a sterile, purified neurotoxin produced by Clostridium botulinum. This organism produces 8 antigenically distinct toxins, 7 of which are neuroparalytic agents. The toxin exerts its paralytic action by binding to presynaptic cholinergic nerve terminals and inhibiting exocytosis and release of acetylcholine. When injected into the muscle at therapeutic doses, botulinum toxin type A (Botox; Allergan Inc, Irvine, California) produces a chemical denervation of the muscle resulting in a localized reduction in muscle activity that generally lasts for a period of 2 to 4 months.
Botox has proven to be an effective clinical treatment for a variety of conditions including blepharospasm, hemifacial spasm, torticollis, strabismus, spasmodic dysphonia, synkinesis, and facial rejuvenation.1 The efficacy of Botox varies from patient to patient, which may be related to individual patient factors, the quantity of toxin per vial (10% variability per 100–mouse unit [MU] vial),2 or the method of reconstitution and storage of the agent. Botox is approved by the US Food and Drug Administration (FDA) for clinical use in the United States, and is available as 100-MU vials of lyophilized C botulinum toxin type A. Each 1 MU corresponds to the calculated median lethal intraperitoneal injection dose (LD50) in mice.3,4 When patients are injected intramuscularly at a therapeutic dose, Botox is not expected to be present in the peripheral blood at measurable levels and therefore should not result in any systemic overt distant effects.
Ever since Botox has been in clinical use, methods of reconstitution and storage of this expensive product have been of great interest. The FDA-approved product labeling recommends intramuscular administration within 4 hours of reconstitution.5 Because a single 100-MU vial may be sufficient to treat up to 5 or 6 patients for forehead rhytids, if administration is limited to a 4-hour window, 2 issues arise: (1) wasting of this expensive product if multiple patients cannot simultaneously accommodate this very restricted time interval; and (2) inconvenience to patients who are faced with limited or restricted intervals to receive treatment.
Only a small quantity of Botox is needed for the injection of any given region on a given patient (eg, forehead rhytids require 12-20 MU; glabellar rhytids, 20-28 MU; lateral periorbital rhytids, 8-24 MU), yet the product is supplied in 100-MU vials. If product recommendations regarding usage within 4 hours of reconstitution were strictly followed, then a substantial quantity of the Botox would likely be discarded if unused (wasting), unless patients are grouped (inconvenience). Consequently, many physicians have adopted the practice of storing (either freezing or refrigerating) the unused reconstituted Botox for later use. To date, this practice has been supported largely by anecdotal observations that the stored reconstituted Botox did not appear to show any noticeable decrease in the clinical effect in human subjects.6- 8
However, a few studies have been performed in an effort to justify the longer storage approach. These studies compared the potency of fresh reconstituted Botox with that of Botox that was reconstituted and then stored (refrigerated or frozen) prior to its use. In 1993, using a mouse lethality bioassay, Gartlan and Hoffman6 concluded that there was approximately a 70% decrease in potency in mice after 2 weeks of freezer storage (−70°C). Sloop et al9 later tested 8 human subjects using electromyographic (EMG)measurements of extensor digitorum brevis activity in the right hand, injected with fresh Botox, compared with the left hand, which was injected with Botox that was either frozen (−20°C) or refrigerated (4°C) for 2 weeks prior to injection. They did not find any difference in paralysis for up to 2 weeks after injection (the duration of their study). In a 1996 publication, Garcia and Fulton10 concluded that 30-day-old Botox produced the same loss of muscle tone in the corrugators, crow's-feet, and frontalis muscles of human subjects as did freshly mixed Botox, but their study was not blinded, and the percentage of denervation was estimated from observation only. To our knowledge, there have been no human studies that examined the impact of duration of storage on facial muscle denervation in a controlled, blinded, structured, statistically meaningful way.
The objective of this study was to address some of the issues not addressed in other articles by testing the clinical efficacy in human subjects of fresh Botox compared with Botox that was reconstituted and then refrigerated or frozen for a 2-week period prior to use. We used a prospective, double-blinded, randomized controlled trial with a side-to-side comparison over a 4-month period.
This study was reviewed and approved by the institutional review board of the University of Pittsburgh Medical Center. Forty subjects were recruited for study participation using announcements posted throughout the University of Pittsburgh Medical Center seeking volunteers for a study of the effect of Botox on forehead wrinkles. Volunteers ranging in age from 21 to 65 years were considered for inclusion. All female participants of childbearing age were tested with a urine pregnancy test to exclude pregnant women from the study. Other exclusion criteria included a history of facial paralysis or weakness from any cause, any history of neuromuscular disorders, any unstable medical condition, known immunodeficiencies, any prior administration of Botox for facial rhytids, and advance knowledge of inability to attend scheduled study follow-up visits.
Exactly 2 weeks prior to the day of administration of Botox to the study participants, four 100-MU vials of Botox were thawed. Each vial was separately diluted with 2.5 mL of preservative-free saline (0.9% isotonic sodium chloride) to yield a final concentration of 4 MU/0.1 mL. The dilution was carried out under a negative pressure laboratory hood to minimize the possibility of bacterial contamination in the product to be stored. All 4 vials were then returned to their original product boxes, which were labeled and taped shut to prevent inadvertent use during the storage period. All 4 vials were subsequently stored for 2 weeks: 2 in a 4°C office refrigerator, and the other 2 in a −20°C office freezer.
During the 2-week storage period, no effort was made to restrict access to the refrigerator or freezer for other routine supplies, so the refrigerator and freezer doors were opened and closed as often as one might expect during routine office practice.
Two weeks later, on the day of Botox administration to the study participants (day 0), four 100-MU vials of Botox A were freshly reconstituted with saline and divided into forty 1-mL insulin syringes with 10 MU of Botox per syringe. The 2-week-old refrigerated (2 vials) and frozen (2 vials) Botox was retrieved (and thawed, if needed), and each was then divided into 20 insulin syringes with 10 MU of Botox per syringe.
All syringes were labeled with a unique number. One of us (R.J.C.) paired each syringe of freshly reconstituted Botox (control) with a syringe of either frozen or refrigerated 2-week-old Botox (experimental). This investigator labeled each pair of coded syringes with a left or right designation to ensure an even distribution of 10 left-sided controls and 10 right-sided controls for each experimental group (frozen vs refrigerated). These designations were recorded in a data key, which was locked in storage and not unblinded until the completion of the study. Each syringe pairing was then sealed in an envelope. Whether a coded syringe contained fresh Botox, refrigerator-stored Botox, or freezer-stored Botox was unknown to the injecting physician (G.S.G.).
Prior to the administration of Botox, subjects were photographed using a standard frontal view with the frontalis muscle at rest and during maximum contraction. A fine-tipped marking pen was then used to mark 2 points on each side of the forehead at rest, in the midpupillary line—one 15 mm and the other 35 mm above the superior margin of the brow. Frontalis muscle movement on each side of the forehead was quantified and recorded by using a caliper to measure the ability of each subject to collapse the distance between the 2 points 20 mm apart during maximum forehead contraction (raising the brow). The forehead movement was determined by measuring the distance between the 2 points at maximum contraction and subtracting that measurement from 20 mm (the distance between the 2 marked points at rest) (Figure 1).
One of us (G.S.G.) then randomly chose a labeled pair of enveloped syringes for injection, recorded which sample each subject received on which side, and sealed this key in an envelope that remained sealed until the end of the study. All investigators and subjects were blinded to which experimental preparation (frozen or refrigerated) was used and which side was administered the control vs experimental preparation for each subject. This blinding technique helped to keep the forehead measurements and subjective questionnaires unbiased and also enabled each subject to be his or her own control. Since one side of each subject's forehead received fresh reconstituted Botox and the other side received a stored Botox preparation, the 40 participants were divided into 2 study groups with 20 subjects per group: fresh vs frozen and fresh vs refrigerated groups.
Each patient received 4 injections (8 MU) of Botox to the frontalis muscle on each side of the forehead. The 4 injections were distributed in a grid pattern between the brow and hairline (Figure 2), staying at least 1.0 to 1.5 cm above the brow to minimize the risk of iatrogenic brow ptosis. Injections were constrained to the space above the brow to avoid the midline forehead between the brows and thus maintain clear separation between the control and experimental injection areas.
Measurements of treatment efficacy and standardized photographs were taken at each study follow-up visit after initial treatment: 2 weeks, 1 month, 2 months, 3 months, and 4 months (Figure 3). At each follow-up visit, photographs and measurements of frontalis muscle contraction were performed and results recorded in the same way as they were prior to injection on day 0. The photographs taken at repose and at maximum contraction were taken using a standard frontal view. Participants were also given a questionnaire to complete at each follow-up visit to provide a subjective assessment of their forehead rhytids and forehead movement and to specifically identify any subjective impression of right vs left forehead movement asymmetry.
In the frozen experimental group, there were 19 women and 1 man with a mean age of 42.7 years (age range, 27-51 years). In the refrigerated experimental group, there were 15 women and 5 men with a mean age of 41.9 years (age range, 23-56 years). Subjects were injected on day 0, and follow-up evaluations were performed at 2 weeks, 1 month, 2 months, 3 months, and 4 months after injection.
One subject in the refrigerated experimental group missed the last 2 follow-up sessions. Two subjects in the frozen experimental group missed 1 follow-up session each (1 at 2 months, 1 at 3 months) but presented for all other follow-up evaluations. Of a total of 200 potential evaluations (40 participants, 5 follow-up evaluations each), only 4 evaluations were missed through the duration of this study.
Of the preinjection measurements in the refrigerated group, 9 subjects had symmetric frontalis muscle contraction, 9 had a 0.5-mm asymmetry, and 2 had a 1.0-mm asymmetry in the measurements. Of the preinjection measurements in the frozen group, 7 subjects had symmetric frontalis muscle contraction, 11 had a 0.5-mm asymmetry, and 2 had a 1.0-mm asymmetry in the measurements. The average pretreatment forehead movement for the frozen group was 5.9 mm, and the average pretreatment forehead movement for the refrigerated group was 6.7 mm. The subjects were randomly assigned to which side received the control vs the experimental Botox.
Frontalis muscle contraction was measured on each side of each participant at each follow-up visit over the course of 4 months. By using each subject as his or her own control, we controlled for individual factors in each subject. Comparisons of the control and experimental sides revealed that the measurements were consistently equal or not more than 0.5- to 1.0-mm different between sides (Table 1 and Table 2) in all but 1 subject, in whom 1.5-mm differences were noted at the 2-week and 1-month follow-ups. In that subject, more movement was measured on the fresh side than on side that received the 2-week frozen Botox.
Differences in measured forehead movement in subjects who received fresh vs frozen or fresh vs refrigerated Botox were analyzed using a paired t test, and differences were considered significant at P < .05. No significant difference was found in the onset or duration of action between the fresh and refrigerated Botox (Figure 4) or between the fresh and frozen Botox (Figure 5). Furthermore, no significant difference in measured movement between the 2 sides was found in either the fresh vs frozen sides or the fresh vs refrigerated sides at any follow-up measurement through the duration of follow-up in this study.
In the review of questionnaires from those who received frozen Botox on 1 side, 4 subjects reported a perceived difference in movement at a total of 5 follow-up evaluations (1 subject on 2 follow-ups). Four of those thought that there was more movement on the control side. In 2 of these cases, there was no measurable difference; in 2 cases, more movement was measured on the perceived side (control); and in 1 case, more movement was measured on the opposite side (control more than frozen experimental). In questionnaires reviewed from those who received refrigerated Botox, 3 subjects felt that there was more movement on the control side and 3 on the refrigerated experimental side. In 2 of those (1 from each group), there was no measured difference. In 2 who thought the refrigerated experimental side had more movement, there was indeed 0.5 mm more movement measured on that side. In the other 2 who thought there might be more movement on the control side, 1 had 0.5 mm more movement on that side, and the other had 1.0 mm more measured contraction on the refrigerated experimental side.
All subjects had a gradual return of muscle function over the 4-month follow-up period. Not all subjects returned to baseline muscle movement in 4 months. A paired t test analysis of the data collected did not find a significant difference in efficacy between the fresh and frozen or between the fresh and refrigerated Botox.
There were no complications or adverse effects reported from the administration of Botox in this study.
Since receiving FDA approval for the treatment of glabellar rhytids, Botox injection has become one of the most popular nonsurgical cosmetic treatments for the aging face. Furthermore, it has a multitude of noncosmetic applications for such conditions as blepharospasm, hemifacial spasm, torticollis, strabismus, laryngeal dystonia, facial synkinesis, Frey syndrome, and other conditions.
The manufacturer of Botox5 recommends that the entire contents of a given vial be used within 4 hours of reconstitution because of concerns relating to loss of potency of the neurotoxin beyond that time period. Regardless of the application, therefore, all practitioners face the same dilemma regarding storage and later use of this expensive product. For the practitioner, the implications of the 4-hour time window are 2-fold: either unused product must be discarded, or efforts must be made to cluster patients into this narrow time interval. As a result, many clinicians have adopted the practice of storing leftover Botox for later use. The viability of this practice is based largely on anecdotal support evidence. Not surprisingly, these issues have sparked interest and research into the effect of cold storage and later use on the potency of Botox.
In 1993, Gartlan and Hoffman6 performed an LD50 mouse bioassay (with intraperitoneal injections of Botox) comparing the substance's potency after refrigeration and freezer storage for 2 weeks.6 Their bioassay showed a 70% loss of potency after storage at –20°C for 2 weeks from the time of reconstitution. However, despite these findings, the mouse LD50 bioassay does not seem to correlate with clinical observations of the potency of Botox used on human muscle, which appears to show no significant reduction in potency after freezing.
The clinical observations in humans have been substantiated by Sloop et al,9 who administered freshly reconstituted Botox to the right extensor digitorum brevis muscle in human subjects to produce a 50% muscle paralysis as a control and compared its efficacy with that of Botox stored for 2 weeks, either frozen or refrigerated, before injection into the left extensor digitorum brevis muscle (each subject thus acting as his or her own control). An EMG comparison at 6 time points (days 2, 4, 6, 9, 11, and 13) showed no statistical difference between each experimental group and its paired controls in the percentage of muscle paralysis measured, thus establishing that storage of Botox for 2 weeks did not affect its ability to produce muscle paralysis in human subjects. However, each experimental group had only 4 subjects, thus limiting the power of the study, and there was no comparative evaluation of duration of effect because muscle paralysis was not measured beyond 13 days. Nonetheless, Sloop et al9 provided some preliminary evidence that the efficacy of Botox in producing muscle paralysis might not be affected by cold storage for 2 weeks.
Hexsel et al11 reported that in the treatment of glabellar frown lines, there was no statistical difference between Botox refrigerated for up to 6 weeks compared with Botox refrigerated for 1 day. Strengths of the study were that it was a prospective, double-blind, randomized trial with human subjects. However, since the freshest of the 2 Botox preparations had already exceeded the 4-hour window that the manufacturer recommends, the data presented could only draw the conclusion that 6-week-old refrigerated Botox was not statistically different from 1-day-old refrigerated Botox. Furthermore, subjects did not serve as their own controls in this study, and the statistical analysis was based on a subjective evaluation of photographs by 6 evaluators using a nonvalidated visual grading scale.
Jabor et al12 performed a study testing frozen Botox that was up to 12 weeks old, using EMG to quantify muscle paralysis in the auricular muscle of rabbits. The 4 groups included fresh Botox (group 1), 2-week-old Botox (group 2), 6-week-old Botox (group 3), and 12-week-old Botox (group 4), with 5 rabbits per group. The conclusion was that the 6-week-old and 12-week-old Botox had a shorter duration of efficacy, but that the 2-week-old Botox did not show any statistical difference from fresh Botox. However, this was an animal study and therefore might not correlate to humans. Also, instead of having each rabbit serving as its own control, which would have been very feasible, group 1 acted as the control group for all other groups. In addition, parameters for comparing groups 2, 3, and 4 were inconsistent. The 2-week-old Botox underwent only 1 freeze-thaw cycle, whereas the 6-week-old Botox underwent 2 cycles, and the 12-week-old Botox underwent 3 cycles. There has been conjecture that the freeze-thaw cycle may cause degradation of Botox, and multiple cycles may cause more degradation of the protein. Thus, this additional variable might have confounded the results. Finally, with such a small sample size (5 rabbits per group), the loss of 1 rabbit would grossly distort the data and the results of any statistical analysis. The mean EMG amplitudes of the 4 groups before Botox treatment (reported by Jabor et al12 as numbers only, with unspecified units) ranged from 7.45 to 11.95, which means that a single EMG amplitude value could have an even wider range. For example, in group 3, the 6-week-old Botox group, the mean pretreatment EMG amplitude was 7.45, but after week 6, 1 rabbit was killed, and the week 8 mean EMG amplitude was 8.51. It is unlikely that the mean EMG amplitude after Botox treatment would be greater than the pretreatment mean.
Recently, Thomas and Siupsinskiene13 reported an open-label crossover study comparing the use of fresh vs frozen-stored Botox in the treatment of 43 patients with laryngeal dystonia. The mean duration of storage for the frozen Botox was 2.4 weeks, with a maximum duration of 8 weeks. The researchers found no difference in duration of action (based on number of weeks without spasm) or efficacy based on a self-rated satisfaction scale. Again, issues of subjectivity arise.
The unique attribute of our study is that 40 subjects each served as their own control and were studied prospectively. Participants and investigators were all blinded to which side of the forehead received the control or experimental Botox. Objective measurements of forehead movement (frontalis muscle contraction) were used on both sides of the forehead, and subjects were observed for 4 months after treatment. Of a total of 200 follow-up measurements scheduled—100 follow-up evaluations for each experimental group (20 subjects each group, 5 follow-up assessments each)—only 4 evaluations could not be completed owing to absenteeism, thus greatly enhancing the power of this study.
In virtually all cases at all follow-up sessions, measurements between the 2 sides differed by no more than 1.0 mm—a difference that is likely of no clinical significance, and a difference that has been seen from side to side on many occasions by any clinician who uses fresh Botox in the treatment of transverse forehead rhytids. For that matter, it is for this very reason (to minimize side to side visible discrepancies) that the pattern of distribution of injections across the forehead is to some degree individualized for any given patient.
In our study, there was no significant difference between subjects who received fresh Botox or 2-week frozen or refrigerated stored Botox with respect to either onset of action, forehead movement, or duration of action through the 4-month study period. Because each experimental preparation was thawed once for reconstitution, and the frozen-stored Botox was thawed a second time on the day of administration, we cannot draw any conclusions regarding the effect of multiple freeze-thaw cycles beyond this. Nonetheless, with typical doses used for facial cosmetic applications, one can generally treat only 2 or perhaps 3 patients from a single 100-MU vial, thereby limiting the number of freeze-thaw cycles almost by default. Furthermore, we cannot draw any conclusions regarding a storage interval in excess of 14 days.
So must Botox really be used within a 4-hour window of reconstitution? Patients who are receiving reconstituted Botox after cold storage should have a reasonable expectation that the effects of the toxin will not be inferior in duration of effect or potency compared with those of freshly reconstituted toxin. There is growing scientific evidence that in fact the product can be stored for some time (frozen or refrigerated) and then used without adverse consequence. While there may never be a human study that is perfectly objective, blinded, and controlled that indicates exactly how long that period of storage might be, we believe that our study lends important credence to the notion that Botox may be stored for at least 2 weeks and then used without statistically or clinically significant loss of potency or duration of effect.
Correspondence: Grant S. Gillman, MD, FRCSC, 5200 Centre Ave, Ste 211, Pittsburgh, PA 15232 (firstname.lastname@example.org).
Accepted for Publication: January 6, 2008.
Author Contributions: All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Yang, Chiu, and Gillman. Acquisition of data: Yang, Chiu, and Gillman. Analysis and interpretation of data: Yang and Chiu. Drafting of the manuscript: Yang, Chiu, and Gillman. Critical revision of the manuscript for important intellectual content: Yang, Chiu, and Gillman. Statistical analysis: Yang. Obtained funding: Yang. Administrative, technical, and material support: Yang, Chiu, and Gillman. Study supervision: Gillman.
Financial Disclosure: None reported.
Funding/Support: This study was supported by the Leslie Bernstein Resident Research Grant (2003) from the American Academy of Facial Plastic and Reconstructive Surgery (Dr Yang).
Role of the Sponsor: The funding organization did not participate in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.
Previous Presentation: This article was presented at the Annual Scientific Meeting of the American Academy of Facial Plastic and Reconstructive Surgery; September 23, 2004; New York, New York.
Additional Contributions: Sandy Shen assisted with subject enrollment and follow-up; Margaret Hung assisted in statistical analysis.