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PubMed2004

Chlorhexidine anaphylaxis: case report and review of the literature.

Chlorhexidine is a widely used antiseptic and disinfectant. Compared to its ubiquitous use in medical and non-medical environments, the sensitization rate seems to be low. Multivarious hypersensitivity reactions to the agent have been reported, including delayed hypersensitivity reactions such as contact dermatitis, fixed drug eruptions and photosensitivity reactions. An increasing number of immediate-type allergies such as contact urticaria, occupational asthma and anaphylactic shock have been reported. In the case report, we describe anaphylaxis due to topical skin application of chlorhexidine, confirmed by skin testing and sulfidoleukotriene stimulation test (CAST(R): cellular antigen stimulation test). The potential risk of anaphylactic reactions due to the application of chlorhexidine is well known, especially that application to mucous membranes can cause anaphylactic reactions and was therefore discouraged. The use of chlorhexidine at a 0.05% concentration on wounds and intact skin was so far thought to be safe. Besides our patient, only one other case of severe anaphylactic reaction due to application of chlorhexidine on skin has been reported. Hypersensitivity to chlorhexidine is rare, but its potential to cause anaphylactic shock is probably underestimated. This review should remind all clinicians of an important potential risk of this widely used antiseptic.

Adult↗

PubMed1993

The binding of delmopinol and chlorhexidine to Streptococcus mutans and Actinobacillus actinomycetemcomitans strains with varying degrees of surface hydrophobicity.

This study evaluated the binding of chlorhexidine and the new surface-active anti-plaque agent delmopinol hydrochloride to Streptococcus mutans and Actinobacillus actinomycetemcomitans cells with various cell surface hydrophobicities. The influence of saliva concentration on the binding of these compounds was also investigated. The radiolabeled compounds were incubated with bacteria and the cells were recovered using a centrifugal filtering technique. Delmopinol had higher binding to the hydrophilic variant strains than to the hydrophobic parent strains; chlorhexidine had higher binding to hydrophobic than to hydrophilic A. actinomycetemcomitans strains and higher binding to hydrophilic than to hydrophobic S. mutans strains. The presence of salivary films decreased the binding of both compounds. Both delmopinol and chlorhexidine had stronger affinity to A. actinomycetemcomitans cells than to S. mutans cells. At equimolar concentrations, delmopinol had a lower binding to all strains tested than chlorhexidine. The high reversibility of the delmopinol binding might be related to a higher diffusion rate and solubility compared with that of chlorhexidine. The amphiphilicity of both molecules is an important feature in their retention to S. mutans and A. actinomycetemcomitans strains of varying hydrophobicities and could play an important role in the substantivity of delmopinol or chlorhexidine in the oral cavity.

Aggregatibacter actinomycetemcomitans↗

PubMed2004

In-use efficacy of a chlorhexidine in alcohol surgical rub: a comparative study.

BACKGROUND: Although full surgical scrubs are performed prior to each case on an operating list, optimum regimens for hand cleaning have yet to be determined, and in-use efficacy evaluations are very limited. METHODS: A crossover study was undertaken comparing a chlorhexidine in detergent/alcohol regimen with povidine-iodine detergent scrub, within an orthopaedic operating environment. Depending on the skin asepsis regimen used, five surgical team members scrubbed or rubbed prior to each case for a complete operating list. Bactericidal efficacy was measured using the 'glove-juice' technique before and after hand asepsis, and at the completion of each case. RESULTS: The chlorhexidine regimen caused substantial and sustained reductions in hand bacterial counts (>50-fold prior to case 1) during surgical cases. Application of alcoholic chlorhexidine prior to each subsequent case reduced bacterial counts to the same level as the original scrub. In contrast, the povidine-iodine scrub reduced counts <3-fold prior to the first case and <2-fold in subsequent cases. The chlorhexidine regimen also resulted in persistent bactericidal effects between cases, as counts prior to application of cases 2 and higher were significantly lower than prior to case 1 (>7-fold for case 2 vs case 1). CONCLUSIONS: The chlorhexidine regimen demonstrated excellent bactericidal efficacy throughout an operating list, and was superior to povidine-iodine scrubbing in all aspects. The alcoholic chlorhexidine regimen is simpler and should have wide surgical application.

2-Propanol↗

PubMed1981

Comparison of the bisbiguanide antiseptics alexidine and chlorhexidine. II. Clinical and in vitro staining properties.

A blind cross-over trial was carried out to compare the tooth and tongue staining associated with the use of a 0.035% alexidine and a 0.2% chlorhexidine mouthrinse. Twenty-two volunteers were divided into two groups termed "tea drinkers" and "non-tea drinkers". All volunteers were requested to refrain from oral hygiene measures throughout two 10-day periods when they rinsed twice a day with the preparation randomly allocated for the respective period. During both periods the members of the groups excluded coffee, red wine and port from their diet. The tea drinking group consumed seven cups of tea per day. Tooth and tongue staining was recorded for extent and severity at the end of each period. The amount of stain accumulating in the two groups was similar following the use of chlorhexidine and alexidine. However, for both chlorhexidine and alexidine the extent and severity of tooth and tongue staining were significantly increased in the tea drinking group. An in vitro study of tea staining of perspex blocks exposed twice a day to 0.035% solutions of alexidine or chlorhexidine throughout a 5-day period demonstrated significantly more staining with alexidine when measured spectrophotometrically. Visually however, the differences in the specimens were minimal. Saliva treatment of the perspex did not significantly alter the staining by alexidine or chlorhexidine. The results provide further evidence for a dietary aetiology to the staining associated with cationic antiseptics. However, alexidine at the concentration used offered no advantage in reducing the side effect of staining when compared with chlorhexidine.

Biguanides↗

PubMed1986

Chlorhexidine compared with other locally delivered antimicrobials. A short review.

Based on the association of bacterial plaque with the initiation of chronic gingivitis and progression of chronic periodontitis, chemical antiplaque agents have been employed both in prevention of periodontal disease and its treatment. In supragingival plaque control regimens, chlorhexidine has not been superceded as a chemical anti-plaque agent, although other compounds have been shown to be useful. The local side-effects of chlorhexidine and other cationic antiseptics, however, limit their long-term use for prevention. Extrinsic tooth staining in particular remains the greatest problem. Short-term anti-plaque uses for chlorhexidine include as an adjunct to mechanical cleaning in the initial oral hygiene phase of treatment, in situations where mechanical oral hygiene is difficult, including postsurgery, intermaxillary fixation, fixed orthodontic therapy, physically and mentally handicapped individuals, systemic diseases with oral manifestations such as leukaemia. More recent interest in chlorhexidine has resulted from the delivery of compounds subgingivally in the treatment of chronic periodontitis. Such methods have extended the use of chlorhexidine into areas inaccessible to the action of antimicrobial drugs delivered locally by conventional means, such as tooth brushing or mouth rinsing. Available evidence suggests that chlorhexidine may not be as effective as some antimicrobial drugs whose activity is more specific for those organisms considered particularly pathogenic to the periodontal tissues.

Anti-Bacterial Agents↗

PubMed1990

A clinical and microbiological comparison of the effects of water and 0.02% chlorhexidine as coolants during ultrasonic scaling and root planing.

The maxillary teeth of 10 patients with moderately advanced chronic periodontitis were treated in a split-mouth design study. The baseline examination included plaque and bleeding scores, probing depths and probing attachment levels. 2 sites in each quadrant were selected for dark-field microscopic analysis. Each quadrant was randomly assigned to test or control and instrumented with an ultrasonic scaler using either 0.02% chlorhexidine or water as the coolant. Measurements were repeated 2, 6 and 10 weeks later, together with additional plaque sampling. Ultrasonic instrumentation with either chlorhexidine or water was equally effective in reducing bleeding scores and improving probing attachment levels. 42% of chlorhexidine- and 38.7% of water-treated sites showed gains of 1 mm or more in clinical attachment. Mean reductions in probing depth were similar (0.9 mm chlorhexidine, and 0.8 mm water). At the final examination, the chlorhexidine-treated quadrants had significantly more sites with probing depths in the 1-3 mm category and less in the greater than 3 mm category than the control quadrants (P less than 0.05). Both treatments reduced the microscopic counts of motiles and spirochaetes, resulting in a subgingival microbiota consistent with periodontal health. The results indicate that chlorhexidine has a slight adjunctive effect in the reduction of pocket depth when used as a coolant during ultrasonic root planing for the treatment of chronic periodontitis.

Adult↗

PubMed1990

Efficacy of Listerine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality.

The experimental gingivitis model was used to compare the anti-plaque, anti-gingivitis and anti-microbial efficacies of a phenolic compound (Listerine) and an amine/stannous fluoride mouthwash (Meridol), using a placebo preparation as negative control and a chlorhexidine solution as positive control in a double-blind study. After professional toothcleaning, 36 volunteers performed optimal oral hygiene for a period of 2 weeks. They then ceased all oral hygiene procedures for 21 days during which they rinsed twice daily with 1 of the 4 mouthrinses. After 3 weeks of rinsing, plaque indices remained the lowest in the chlorhexidine group, while subjects using Listerine or Meridol harbored similar indices significantly lower than that of individuals rinsing with the placebo solution. Up to that time, the gingival index scores were equal in all groups except for the chlorhexidine group in which the values only amounted to half of these encountered in the other groups. The plaque vitality scores showed a bactericidal effect in vivo of chlorhexidine during the entire time of experimental gingivitis. In contrast, the data gave no evidence of an antibacterial effect in vivo of Listerine. The efficacy of Meridol to kill micro-organisms was similar to chlorhexidine during the early stages of plaque accumulation and, with time, became insignificant. This study has demonstrated that chlorhexidine was superior to Listerine and Meridol in its ability to maintain low plaque scores and gingival health during this 3-week period of no mechanical oral hygiene. Moreover, it was also shown that Meridol was as effective as Listerine in reducing plaque accumulation and, in contrast to Listerine, possessed a remarkable but transient antibacterial effect in vivo.

Adult↗

PubMed1992

Efficacy of Listerine, Meridol and chlorhexidine mouthrinses as supplements to regular tooth cleaning measures.

The anti-plaque, anti-gingivitis and anti-microbial efficacies of a phenolic compound (Listerine) and 2 different amine/stannous fluoride mouthwashes (Meridol I, II) were compared when these solutions were used in addition to usual tooth cleaning. A placebo preparation was utilized as a negative control and a chlorhexidine solution as a positive control in this double-blind study. After professional tooth cleaning, 49 volunteers continued their habitual, self-performed and non-supervised oral hygiene for a period of 2 weeks, in order to have a more standard baseline. At day 0, they began to rinse twice daily with 1 of the 5 mouthwashes. After 3 weeks of rinsing, plaque indices remained the lowest in the chlorhexidine and the Meridol I groups, while subjects using Listerine or Meridol II demonstrated similar indices significantly lower than that of individuals rinsing with the placebo solution. Through this period, the gingival index scores were similar in the Meridol, Listerine and chlorhexidine groups. At day 21, the mean GI scores in the chlorhexidine group were significantly lower than the scores in the placebo group. The plaque vitality scores showed a bacterial effect in vivo of chlorhexidine and, to a lesser extent, of the Meridol solutions. No substantial evidence of an antibacterial effect in vivo was found for Listerine. This study has demonstrated that when mouthrinses are used to supplement habitual mechanical oral hygiene, chlorhexidine remains the most powerful solution. Furthermore, it was also shown that a combination of habitual self-performed and non-supervised oral hygiene with Meridol or Listerine is more beneficial for plaque control than the use of mechanical oral hygiene alone.

Adult↗

PubMed1995

Comparative staining in vitro and plaque inhibitory properties in vivo of 0.12% and 0.2% chlorhexidine mouthrinses.

The mere incorporation of an active ingredient in an oral hygiene product does not necessarily guarantee efficacy. As new formulations appear, it would seem prudent to at least screen for activity by comparison with an established product. The aim of this study was to compare a new 0.12% chlorhexidine containing rinse with a well researched 0.2% chlorhexidine rinse product. The rinses were firstly compared in vitro for dietary staining effects and in vivo for plaque inhibition. Both formulations in vitro produced progressive staining of acrylic specimens with increasing passages through a standard tea solution. The clinical investigation was a 3-treatment, randomised, double blind, crossover, 4-day plaque regrowth study, balanced for 1st and 2nd order residual effects and involving 24 volunteers. From a zero plaque baseline, volunteers suspended oral hygiene and rinsed 2 x per day with the allocated rinse. The chlorhexidine doses were 18 mg and 20 mg per rinse for the 0.12% and 0.2% formulations, respectively. Plaque was scored by area and index on day 5. The results showed that the 2 chlorhexidine rinses were similar in efficacy by comparison with the control rinse. These studies in vitro and in vivo indicate that the chlorhexidine in the new preparation is available and active and the product could find use in those preventive applications suggested for other chlorhexidine formulations.

Acrylic Resins↗

PubMed1995

The comparative tea staining potential of phenolic, chlorhexidine and anti-adhesive mouthrinses.

Staining of teeth and mucous membranes is a well-known side-effect with chlorhexidine mouthrinses in which dietary chromogens play an important rôle. The purpose of this study was to determine whether a co-polymer anti-adhesive agent would prevent staining by a low concentration chlorhexidine solution. Additionally, the possibility that an essential oil/phenolic rinse product may cause staining was investigated. The rinses studied were the anti-adhesive alone and combined with 0.02% chlorhexidine and the essential oil/phenolic rinse. These were positioned against a positive control rise, 0.2% chlorhexidine, and a negative control rinse, water. The study was a single blind 5-treatment, randomised Latin square cross-over design, incorporating balance for carry-over effects. 15 volunteers participated and on Day 1 of each study period were rendered stain free by scaling and polishing of the teeth. Oral hygiene was suspended and 8 x per day subjects rinsed under supervision, firstly with the allocated formulation and then with 10 ml of warm black tea. On Day 4, tooth and tongue staining was scored by area and intensity (colour). A washout period of at least 3 1/2 days was permitted between treatment periods when oral hygiene was resumed. Before the study and during washouts, volunteers practised tongue brushing. Tooth and tongue staining was significantly increased with 0.2% chlorhexidine compared to the essential oil/phenolic rinse which in turn was significantly increased compared to the other 3 rinses. The antiadhesive/chlorhexidine rinse produced no more staining than the anti-adhesive or water rise. However, the parallel plaque regrowth study suggests this inhibition of staining resulted from the vitiation of the chlorhexidine activity by the antiadhesive. The methodology would appear a simple and quick way of assessing the propensity of mouthrinses to cause extrinsic staining.

Anti-Infective Agents, Local↗

PubMed1998

The use of locally delivered chlorhexidine in the treatment of periodontitis. Clinical results.

Since the advent of a nondegradable controlled local delivery of antibiotics in 1979, several second generation systems have been developed. Second generation systems have attempted to improve on the early system. Chlorhexidine has been used effectively for over 30 years as an antiseptic. In the early 1970s, chlorhexidine gluconate was incorporated at 0.2% into mouthrinses in Europe and in 1986 it was incorporated at 0.12% in a mouthrinse in the United States. Since these mouthrinses were effective in reducing the supragingival flora, had a high safety margin, and had no reported bacterial resistance, chlorhexidine offered a therapeutic advantage for a local delivery system. This system was developed and studied. This report will discuss this new biodegradable system containing chlorhexidine gluconate as the active agent (PerioChip). Pharmacokinetics of the system and a review of the multicenter studies in Europe and the United States are discussed. In these randomized clinical trials the chlorhexidine chip has been shown to enhance the effects of scaling and root planing. Chlorhexidine chip in conjunction with scaling and root planing, when compared to scaling and root planing alone, has shown significant improvement in probing pocket depth reduction, probing attachment level and bleeding on probing. This delivery system, in combination with scaling and root planing, has also resulted in significantly more probing depth reductions of 2 mm or more. The system is safe and efficacious. Placement of the chip is usually done in less than 1 min, it requires no retention system, biodegrades, and does not require a follow-up dental appointment.

Administration, Topical↗

PubMed2006

Effectiveness of a chlorhexidine dentifrice in orthodontic patients: a randomized-controlled trial.

This blind and randomized-controlled trial analysed chlorhexidine dentifrices in relation to dental plaque, gingivitis, bleeding, calculus and enamel extrinsic staining development. Volunteers in fixed orthodontic therapy used the following dentifrices: 1100 ppmF, NaF (group A, n=27); experimental, 1100 ppmF, NaF and chlorhexidine 0.95% (group B, n=28); and experimental, chlorhexidine 0.95% (group C, n=28). At baseline, after 6, 12 and 24 weeks, clinical examinations were carried out. The gingivitis, bleeding and plaque data were tested by anova and Tukey's post hoc tests. Stain and calculus data were analysed by Kruskal-Wallis and Dunn's post hoc tests (p<0.05). Plaque, gingivitis and bleeding scores improved in all three groups, but up to the 6 and 12 weeks examination the products containing chlorhexidine were statistically better. The chlorhexidine dentifrices significantly increased the mean of the stain index, although most of the patients did not notice the stains. The calculus index was not significantly modified. In summary, this study suggests that the use of dentifrices containing chlorhexidine seems to be effective for the treatment of gingivitis in orthodontic patients, although the intense motivating contact that the volunteers had with the researchers may have also played a role.

Adolescent↗

PubMed1976

The effect of chlorhexidine mouthrinse on healing after gingivectomy.

The purpose of this investigation was to evaluate the effect of 0.2% chlorhexidine gluconate mouthrinse following gingivectomy on plague under the dressing and on healing. Twenty-eight patients with indications for gingivectomy were selected. Coe-Pak was used as surgical dressing. In addition to unsual home care, the patients rinsed twice daily with chlorhexidine or placebo for 21 d after surgery. The study followed a cross-over double-blind design. Gingival exudate was assessed and Pl I and G I were registered at 7, 14, and 21 d postsurgically. The study indicated that chlorhexidine did not influence the amount of plaque under the dressing, and it was uncertain whether chlorhexidine had any effect on the healing process when the surgical area was covered by Coe-Pak. However, (1) after the surgical dressing was removed, the chlorhexidine maintained plaque scores at the same low level as under the dressing, (2) healing was was promoted when chlorhexidine was used, and (3) the presence of a dressing in one side of the mouth did not prevent the patient from maintaining good oral hygiene.

Adolescent↗

PubMed1985

Importance of teeth and tongue as possible receptor sites for chlorhexidine in relation to its clinical effect.

Teeth have previously been shown not to be of any major importance when quantitatively measuring the retention of chlorhexidine in the oral cavity. Only a few studies have examined the significance of the binding of chlorhexidine to the tongue in relation to its plaque inhibiting effect. The present study was therefore carried out to further examine these possible chlorhexidine receptor sites in the oral cavity in relation to its known antibacterial effect at the teeth surface. Two daily applications of chlorhexidine on the teeth in the right side of the maxilla, against a contralateral water control, both in combination with a sucrose challenge six times daily, showed no significant reduction of plaque formation when compared to an application of sucrose used alone. Two daily applications of chlorhexidine on the tongue, again combined with sucrose challenge, also showed no significant plaque inhibiting effect. The results obtained indicate that neither the teeth nor the tongue seem to be of major importance as receptor sites for chlorhexidine in the oral cavity as related to its observed clinical effect.

Administration, Topical↗

PubMed1997

Effects of chlorhexidine on the bacterial colonization and degradation of dentin and completely demineralized dentin in situ.

The effects of 0.2% chlorhexidine on selected plaque microorganisms were studied in an intraoral dentin caries model. In 8 individuals wearing partial dentures, sound and completely demineralized dentin specimens were placed consecutively in 2 periods of 4 weeks, respectively. Throughout the experimental period, the specimens were treated 2 x daily with 0.2% chlorhexidine; control specimens were treated with water. Plaque accumulation on the specimens was left undisturbed. No protection against demineralization of the dentin or degradation of the dentin collagen by the chlorhexidine treatment was observed. The chlorhexidine treatment did not result in a reduction of the total cultivable flora when compared with the control specimens. A significant reduction of mutans streptococci and total streptococci recovered from completely demineralized dentin treated with chlorhexidine was observed, but the proportions of Actinomyces and lactobacilli were not affected significantly. It is speculated that areas of exposed roots, which are difficult to reach by oral hygiene measurements, such as approximal surfaces, will not be protected by a 0.2% chlorhexidine mouthrinse against the caries process.

Actinomyces↗

PubMed1993

Analytical electron microscopy of chlorhexidine-induced tooth stain in humans: direct evidence for metal-induced stain.

Using analytical electron microscopy, we directly tested the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. Plaque scraped from the teeth of water-treated individuals or chlorhexidine-treated "non-stainers" was non- or lightly-stained and contained low concentrations of sulfur and transition metals. Tooth scrapings from heavily-staining chlorhexidine-treated individuals consisted of distinct unstained and stained regions. The stained regions were organic but were in close proximity to mineralized areas. Enhanced levels of sulfur and transition metals, particularly iron, were found in stained regions, whereas unstained regions contained low sulfur and metal levels similar to the water-treated or non-staining individuals. Excluding decreased mineralization, the major elemental change in heavily-stained plaque was an increase in sulfur, and to a lesser extent, iron and other transition metals. Sulphur and iron levels were directly correlated. Following chlorhexidine treatment with iron supplementation, staining was enhanced, the Fe/S ratio increased, and sulfur and iron remained correlated. These data support the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. The data are consistent with chlorhexidine treatment altering the incorporation into plaque of a natural sulfur-containing organic component of saliva or bacteria. This natural component appears to readily interact with transition metals, particularly iron, producing stained material.

Chlorhexidine↗

PubMed2004

Chlorhexidine induces DNA damage in rat peripheral leukocytes and oral mucosal cells.

OBJECTIVE: Chlorhexidine digluconate is widely used in dental practice for decreasing plaque control, controlling gingivitis and disinfecting root canals. However, the undesirable effects of chlorhexidine digluconate regarding its genotoxicity are conflicting in the literature. Thus, the aim of this study was to investigate the genotoxicity of chlorhexidine digluconate in rat peripheral blood and oral mucosal cells by the single cell gel (comet) assay and micronucleus assay. METHODS: Thirty male Wistar rats were distributed into three groups: negative control; experimental group orally treated with 0.5 ml of 0.12% chlorhexidine digluconate, twice daily, during 8 days; and positive control, which received 4-nitroquinoline 1-oxide at 0.5 g/l by drinking water. RESULTS: A statistically significant increase of DNA damage was observed in leukocytes and oral mucosal cells of the chlorhexidine digluconate treated group, as assessed by the comet assay. However, no increase of micronucleated cells was detected in reticulocytes from peripheral blood cells. CONCLUSIONS: Taken together, the data indicate that chlorhexidine digluconate is able to induce primary DNA damage in leukocytes and in oral mucosal cells, but no chromosome breakage or loss in erythrocytes.

Animals↗

PubMed2000

The effect of brief exposure to sub-therapeutic concentrations of chlorhexidine gluconate on the germ tube formation of oral Candida albicans and its relationship to post-antifungal effect.

OBJECTIVES: Adherence of Candida albicans has been implicated as the first step in the pathogenesis of oral candidosis, and germ tube formation a contributory attribute. Recently, these organisms have also been implicated in persistent apical periodontitis. Chlorhexidine gluconate is by far the commonest antiseptic mouth wash prescribe in dentistry. As the intraoral concentrations of this antiseptic fluctuate considerably due to the dynamics of the oral cavity, the main objective of this study was to investigate the effect of brief exposure to three different sub-therapeutic concentrations of chlorhexidine gluconate (0.005%, 0.0025% and 0.00125%) on the germ tube formation of C. albicans. These findings were then correlated with the chlorhexidine-induced post-antifungal effect (PAFE) values we obtained in a study using the identical organisms and experimental conditions. DESIGN: Ten oral isolates of C. albicans were exposed to three different concentrations of chlorhexidine gluconate for 30 min, the antiseptic removed, and the germ tube formation of these isolates quantified following subsequent incubation in a germ tube inducing medium. The PAFE was evaluated by turbidometric measurement of growth. RESULTS: When compared with the controls, exposure to 0.005%, 0.0025% and 0.00125% chlorhexidine gluconate suppressed the ability to form germ tubes by 81.23% (P < 0.01), 42.74% (P < 0.01) and 9.13% (P > 0.05), respectively, while eliciting a mean PAFE of 9.91 h, 1.65 h and 0.67 h respectively. On regression analysis a significant positive correlation was observed between these two parameters (P < 0.0001; r = 0.7325). CONCLUSIONS: Taken together, these findings imply that short exposure to sub-therapeutic levels of chlorhexidine gluconate may modulate candidal germ tube formation as well as its growth, thereby suppressing its pathogencity in vivo.

Analysis of Variance↗