兽研Frontiers in Veterinary Science01frontiersin.org 类型 原创研究 发表于 2026年10月5日 DOI 10.3389/fvets.2026.1918882 开放获取 编辑：Benito Soto-Blanco，巴西米纳斯吉拉斯联邦大学 审稿：Hilke Oltmanns，德国汉诺威兽医大学；Ujban Hussain，印度那格浦尔Rashtrasant Tukadoji Maharaj大学 *通讯作者：Daniela Siel daniela.siel@umayor.cl；Jessica Bravo jessica.bravo@mail.udp.cl 收稿：2026年6月25日 修回：2026年8月10日 接受：2026年8月24日 发表：2026年10月5日 引用格式：Varas M, Jacobs M, Rojas N, Ramos P, Bruna F, Valenzuela-Barra G, Correa O, Díaz-Parra A, Ahumada M, Maturana G, Martínez I, Abusleme F, Rivera B, Bargsted MO, Siel D and Bravo J (2026) 用于犬皮肤给药的Cryptocarya alba（Peumo）和Laureliopsis philippiana（Tepa）精油干洗香波制剂的稳定性及皮肤安全性评估。Front. Vet. Sci. 13:1918882. doi: 10.3389/fvets.2026.1918882 版权 © 2026 Varas、Jacobs、Rojas、Ramos、Bruna、Valenzuela-Barra、Correa、Díaz-Parra、Ahumada、Maturana、Martínez、Abusleme、Rivera、Bargsted、Siel和Bravo。本文为开放获取文章，依据知识共享署名许可（CC BY）条款发布。允许在其他论坛使用、分发或复制，前提是注明原作者和版权所有者，并按照公认的学术规范引用本刊原始发表文献。不符合这些条款的使用、分发或复制均不被允许。用于犬皮肤给药的Cryptocarya alba（Peumo）和Laureliopsis philippiana（Tepa）精油干洗香波制剂的稳定性及皮肤安全性评估 Martín Varas 1,2，Martina Jacobs 1,2，Nicole Rojas 1，Paola Ramos 1，Flavia Bruna 1,3,4，Gabriela Valenzuela-Barra 1,5，Olosmira Correa 1,5，Antonia Díaz-Parra 1,6，Manuel Ahumada 6，Gabriela Maturana 1，Irene Martínez 2，Francisco Abusleme 7,8，Belén Rivera 8，María Olga Bargsted 7，Daniela Siel 7,9 * 和 Jessica Bravo 1 * 1 智利圣地亚哥，Diego Portales大学医学院生物医学研究中心生物活性天然产物实验室，2 智利圣地亚哥，智利大学物理与数学科学学院化学工程、生物技术与材料系，3 阿根廷门多萨，CONICET CCT Mendoza—国立库约大学（UNCuyo）实验医学与生物学研究所（IMBECU）激素与癌症生物学实验室，4 阿根廷门多萨，国立库约大学（UNCuyo）牙学院牙科研究中心（CIO），5 智利圣地亚哥，智利大学化学与药学院药物科学与技术系，6 智利圣地亚哥，Mayor大学科学、工程与技术学院生物技术学院，7 智利圣地亚哥，Mayor大学医学与健康科学学院兽医学院，8 智利圣地亚哥，Oftaderm兽医诊所，9 智利圣地亚哥，Mayor大学生物医学中心 引言：抗生素耐药性是人和兽医医学中的主要关切，这支持了为伴侣动物开发替代性局部产品的必要性。本研究旨在开发并评估含有Cryptocarya alba和Laureliopsis philippiana精油的干洗香波制剂，作为用于犬皮肤护理和浅表性脓皮病辅助管理的潜在兽医皮肤化妆品平台。方法：使用玉米淀粉、木薯淀粉或其混合物开发了含有C. alba或L. philippiana精油的淀粉基干洗香波原型。评估了硬脂酸镁（MgS）作为功能性辅料的作用，并比较了两种混合方案。对优化后的制剂进行了理化表征、热学、力学和9个月稳定性评估，以及针对假中间葡萄球菌的体外抗菌试验。通过宏观刺激评分、体重监测和组织学分析，在BALB/c小鼠中评估了临床前皮肤安全性。随后，在32只客户拥有的浅表性脓皮病犬中开展了一项探索性、随机、对照、盲法、四臂平行组研究，分配接受SLP、SCA、载体香波或基于氯己定的参考治疗，为期21天。结果：MgS改善了制剂的均匀性、流动性并减少了结块，而所选混合方案提高了粉末的整体性能。
Varas 等，10.3389/fvets.2026.1918882，《 Frontiers in Veterinary Science》02 frontiersin.org 优化后的配方显示出初步的物理稳定性，尽管在储存过程中观察到质地、流动性、团聚倾向和香气发生渐进性变化。体外抗菌活性呈试验方法依赖性：琼脂扩散法未观察到可测量的抑菌圈，而系列稀释法和活菌计数试验显示细菌回收率呈浓度依赖性降低，包括与赋形剂配方相关的效应。小鼠未观察到皮肤刺激的宏观或组织学迹象。在犬中，这些配方在所评估条件下耐受良好。微生物学结果在各组间数值上存在差异但无显著性；在含精油的配方中，SCA 显示出最有利的探索性特征，包括 3/8 只犬培养阴性以及达到非显著细菌负荷的犬比例最高。讨论：这些发现支持将 C. alba 和 L. philippiana 精油掺入淀粉基干香波原型的可行性，并提供了物理稳定性和皮肤耐受性的初步证据。然而，试验方法依赖性的抗菌反应、碱性配方 pH、有限的批次重复以及探索性临床设计，使得无法就临床疗效得出结论。进一步研究应包括配方优化、定量化学稳定性评估、皮肤渗透研究以及更大规模的随机对照临床试验。关键词：精油、Cryptocarya alba、Laureliopsis philippiana、干香波、犬浅表性脓皮病、假中间葡萄球菌 1 引言 脓皮病是犬最常见的健康问题之一，通常由细菌引起，其中假中间葡萄球菌是最常见的病原体(1)。脓皮病的传统治疗包括抗生素(2)，但抗生素滥用现在是细菌耐药的主要原因之一，这是人类和动物健康的主要关切。迫切需要新的抗生素药物来治疗多重耐药细菌(3)。局部药物制剂被推荐用于治疗浅表性脓皮病，即使存在耐甲氧西林菌株(2)。先前的研究探索了犬浅表性脓皮病的替代局部策略。例如，基于环糊精的方案在选定病例中显示出与全身抗生素治疗相当的疗效，突显了局部应用配方减少对全身抗菌药物依赖的潜力(4)。然而，局部疗法的性能取决于多种配方相关因素，包括活性化合物的浓度、表面活性剂的使用、pH、施用频率和接触时间(5, 6)。推荐每周施用两至三次，接触时间 5 至 10 分钟，但研究表明长期治疗可能破坏真皮的正常保护功能，这在先前有炎症皮肤的患者中尤为重要(6)。植物精油是含有萜烯、萜类化合物和苯基萜烯以及其他生物活性化合物的复杂混合物(7, 8)。植物合成这些次级代谢产物作为防御病原体的机制，并且由于其抗氧化、抗炎、抗菌和抗真菌特性(7–10)，精油已被研究用于制药行业(11)。Cryptocarya alba（Peumo）和 Laureliopsis philippiana（Tepa）是智利原产的两种树种，马普切人曾用其治疗肝病、风湿病和伤口(12, 13)。从 C. alba 和 L. philippiana 获得的精油富含 1,8-桉叶素（也称为桉油精）、芳樟醇和黄樟醚等化合物(13, 14)，其抗菌活性已被报道对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和肺炎克雷伯菌等有效(13, 14)。对于 intended 重复使用的兽用局部制剂，在临床使用前必须进行体内评估以确定安全性和生物学性能。小鼠模型广泛用于评估局部皮肤耐受性，包括红斑、水肿、组织学改变和潜在的全身效应，而犬作为目标物种的研究允许在真实皮肤病学条件下初步评估临床和微生物学反应(15–22)。本研究假设 C. alba 和 L. philippiana 精油可掺入淀粉基干香波中，具有可接受的物理稳定性和局部皮肤耐受性，并且所得配方可在患有浅表性脓皮病的犬中产生可测量的微生物学反应。因此，主要目标是开发和表征载有精油的干香波 缩写：CA，Cryptocarya alba；CA-EO，Cryptocarya alba 精油；EO，精油；LP，Laureliopsis philippiana；LP-EO，Laureliopsis philippiana 精油；MgS，硬脂酸镁；SCA，载有 CA-EO 的香波配方；SLES，月桂醇聚醚硫酸酯钠；SLP，载有 LP-EO 的香波配方；SLP1，含玉米淀粉和 LP-EO 的配方；SLP1-MgS，含玉米淀粉、MgS 和 LP-EO 的配方；SLP2，含木薯淀粉和 LP-EO 的配方；SLP2-MgS，含木薯淀粉、MgS 和 LP-EO 的配方；SLP3，含混合淀粉来源和 LP-EO 的配方；SLP3-MgS，含混合淀粉来源、MgS 和 LP-EO 的配方；SVE，香波赋形剂。
Varas等人 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science03frontiersin.org 原型，评估其临床前皮肤安全性，并评价其在患有浅表性脓皮病的客户自有犬中的微生物学性能。2 材料与方法 本研究的实验流程总结于图1。简言之，方法学包括五个连续阶段：植物采样与精油提取、干香波配方开发、理化表征、小鼠临床前安全性测试以及犬临床评价。每个阶段均旨在逐步评估配方的可行性、稳定性、安全性和微生物学性能。2.1 采样与提取 于春季（2024年11月）花期结束、果实形成之前，在智利首都大区兰帕市奇卡乌马（南纬33°12′3.8808′′，西经70°58′8.1156′′）采集C. alba（CA）新鲜叶片。同样，于2024年10月在智利洛斯里奥斯大区瓦尔迪维亚市洛斯佩利内斯（南纬39°44′42.0′′，西经73°21′25.2′′）花期采集L. philippiana（LP）新鲜叶片。为两个物种制备了凭证标本。CA（SQF 22901）存放于智利大学化学与药学学院植物标本馆，LP（VALD 3560）存放于智利南方大学植物标本馆。植物材料于采集当日或此后24小时内处理用于提取。两个物种的精油提取均在采集后立即使用10 L Clevenger型装置通过水蒸气蒸馏进行。该过程分批进行，直至全部植物材料蒸馏完毕（两种情况下每批1小时）。所得非均相混合物使用分液漏斗分离以回收精油。水溶胶弃去。由此获得两种精油：CA精油（CA-EO）和LP精油（LP-EO）。这些精油的化学特征此前已由Jacobs等人（23）通过气相色谱-质谱联用（GC-MS）表征。在该分析中，两种油共检测到62种挥发性化合物。CA-EO显示出更复杂的特征，鉴定出43种化合物，其中桧烯和桉叶油素为主要成分，分别占总色谱面积的13.95%和12.05%。相比之下，LP-EO鉴定出20种化合物，以芳樟醇、桉叶油素和黄樟脑为主，分别占总色谱面积的37.91%、24.54%和11.42%。这些发现证实CA-EO和LP-EO具有明显不同的植物化学特征，这可能有助于其生物学和抗菌特性的差异。CA-EO和LP-EO对假中间葡萄球菌的体外抗菌活性此前也已由Jacobs等人（23）报道。在该研究中，两种精油针对来自脓皮病犬的12株S. pseudintermedius分离株以及参考菌株S. pseudintermedius ATCC 49444，采用琼脂扩散法和最低抑菌浓度（MIC）试验进行了评价。两种油均显示出中等抗菌活性，抑菌圈范围为4.92至10.80 mm。CA-EO的平均抑菌直径高于LP-EO，平均值分别为9.09 mm和6.76 mm，分别相当于参考消毒剂氯己定 observed 活性的37%和28%。对CA-EO最敏感的菌株为SP9，对LP-EO最敏感的菌株为SP13，抑菌圈分别为10.38和10.80 mm。两种油的MIC值一般≥4.096 mg mL−1，但LP-EO对菌株SP12的MIC为2.048 mg mL−1。这些结果支持两种精油的抗菌潜力。图1 本研究的实验流程。方法学顺序包括植物采样与精油（EO）提取、配方开发、理化表征、BALB/c小鼠临床前安全性测试以及犬临床评价。CA-EO：Cryptocarya alba精油；LP-EO：Laureliopsis philippiana精油；MgS：硬脂酸镁；SCA：含C. alba精油的干香波；SLP：含L. philippiana精油的干香波；SVE：载体配方；FTIR：傅里叶变换红外光谱；DSC：差示扫描量热法；SEM：扫描电子显微镜；CFU：菌落形成单位。
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，04frontiersin.org，对假中间葡萄球菌具有活性，尽管活性低于氯己定。2.2 配方与均匀性试验 为评估均匀性，参照 AGRANA STARCH 1 和 AkzoNobel Surface Chemistry 的商业干香波配方示例，设计制备了六种含 LP-EO 的香波配方（SLP），每份 10 g 样品。为评估均匀性，参照商业产品（AGRANA – MAISITA 9040 和 Zephyrous Dry Shampoo 2,906–100）并参考先前关于干草药粉香波配方的工作（24），制备了六种含 LP-EO 的香波配方（SLP），每份 10 g 样品。测试了三种淀粉：玉米淀粉、木薯淀粉及其混合物。选择硬脂酸镁是因为其已在固体药物和化妆品配方中确立使用，且据报道可影响粉末流动性、颗粒内聚性和团聚性。这些特性被认为对改善亲脂性精油在淀粉基质中的掺入和分布尤为重要。为评估其功能贡献，制备了除 MgS 外其他方面可比的含与不含 MgS 的配方（25–27）。配方按所用淀粉分组，每组再根据是否添加 MgS 分为两组，共得到 6 种配方：基于玉米淀粉不含 MgS 的配方（SLP1）和含 MgS 的配方（SLP1-MgS），基于木薯淀粉不含 MgS 的配方（SLP2）和含 MgS 的配方（SLP2-MgS），基于玉米淀粉和木薯淀粉混合物不含 MgS 的配方（SLP3）和含 MgS 的配方（SLP3-MgS）。为目视评估精油分散情况，预先将 LP-EO 与油溶性染料苏丹红 B（Merck，智利）混合。在适用时，将淀粉与 MgS 混合，然后加入用苏丹红 B 着色的 LP-EO。混合物在 Incu-Shaker™ Mini 中以 200 rpm 振摇 18 h，用研钵和研杵研磨以打散团聚体。随后掺入十二烷基聚氧乙烯醚硫酸钠（SLES）1 AGRANA STARCH。含 MAISITA 9040 和 TAPIOCA NATURAL 的非气雾干香波。见：https://www.agrana.com/downloadcenter?tx_thagranadownloadcenter_downloadcenter%5Baction%5D=download&tx_thagranadownloadcenter_downloadcenter%5Bcontroller%5D=Downloadcenter&tx_thagranadownloadcenter_downloadcenter%5Bdownload%5D=4856&type=97687851&cHash=6f68fc458981b7bde90b514c1f5f7bcf 2 AkzoNobel Surface Chemistry。Zephyrous Dry Shampoo。见：https://www.essentialingredients.com/pdf/AkzoNobel%20Zephyrous%20Dry%20Shampoo%20%282906-100%29.pdf 作为阴离子表面活性剂，以改善疏水性精油在粉末基质中的润湿和分散，并提供与配方作为局部干香波预期用途一致的清洁功能（28）。然后将混合物再振摇 2 h（图 2）。此混合方法称为方法 A。所得粉末采用基于七项感官和物理参数的定性量表进行评估（表 1），包括均匀性、质地、颜色、流动性和气味，按 1 至 5 的定性量表评分，该量表根据药物和化妆品配方所描述的一般感官和物理评价标准设计（28, 29）。选择视觉均匀性更好且结块倾向更低的样品进行混合试验。2.3 混合试验 本阶段旨在确定最能最大化配方均匀性的高效混合方法。评估了两种混合方法，考虑到 MgS 在粉末混合物中的分布及其对流动相关性质的影响可能取决于混合条件和组分加入顺序（26, 27）。方法 A 见第 2.2 节和图 2。对于方法 B（图 3），先将淀粉源与精油混合，然后加入 MgS，最后加入 SLES，中间进行研磨步骤以增强分散。仅对含 MgS 的配方进行混合试验，即 SLP1-MgS、SLP2-MgS 和 SLP3-MgS。在此阶段，精油浓度降至 2%，染料浓度减半，SLES 比例增加。在 Incu-Shaker™ Mini 中以 200 rpm 进行混合，必要时辅以手动研磨以打散团聚体。采用表 1 所述的相同感官量表评估配方。选择实现最高组分整合度和最佳粉末流动性的方法制备用于本研究后续阶段的原型。最终原型的选择基于均匀性、粉末流动性、结块倾向、精油掺入、操作特性和后续物理稳定性的总体平衡。2.3.1 评分量表 为评估组分整合度和整体配方均匀性，采用包含七项物理和 图 2 用于均匀性试验的配方成分混合程序。此程序称为方法 A。含 CA-EO 的配方类似应用相同程序。CA-EO：Cryptocarya alba 精油。LP-EO：Laureliopsis philippiana 精油。MgS：硬脂酸镁。SLES：十二烷基聚氧乙烯醚硫酸钠。
Varas 等，10.3389/fvets.2026.1918882，《 Frontiers in Veterinary Science》，05，frontiersin.org。在均匀性、混合、热稳定性和长期稳定性试验中采用了感官参数评价。每个参数根据表1中呈现的描述符按1至5分评分，这些描述符基于洗发水和化妆品配方研究中通常考虑的评估标准而建立（24、28、30）。评估由一名非盲评估者完成，该评估者即为执行实验程序的研究人员。每个样品或实验条件在相应的评估时间点仅评估一次；因此，未进行独立的重复感官评估。 consequently，评估者间一致性和评估者内一致性分析不适用。2.4 配方的初步理化表征。对于宏观理化评价，使用混合方法A制备玉米淀粉/MgS配方，得到三个样品：SLP（载LP配方）、SCA（载CA配方）和SVE（赋形剂配方）。测量在10 g样品上进行。对于本探索性配方研究，每个最终原型（SLP、SCA和SVE）均作为单一批次制备。未生产独立制备的重复批次；因此，未评估批间重现性。试验包括pH测量、热稳定性。表1 均匀性、混合、热稳定性和长期稳定性试验的评估标准。评分 视觉均匀性 无结块 颜色均匀性 触感质地 流动性 团聚倾向 1 不均匀混合物。油分布不良。大而多的结块。颜色分布不规则，存在明显界限的彩色和无色区域。极粗糙或致密，难以处理。容器倾斜后混合物不流动。完全压实。2 轻微不均匀。混合物看似均匀。分散的中等大小结块。颜色分布稍均匀，饱和度差异少。粗糙、高黏聚性颗粒。轻微流动，需强烈搅拌才能移动。持续结块。可见成团。3 基本均匀。质地差异轻微可见。小结块。易崩解。颜色基本均匀。柔软。轻微粗糙。容器倾斜时，无搅拌下混合物缓慢流动。搅拌时可逆成团。4 均匀。未见不规则区域。未见结块。表面光滑。颜色均匀且稳定。未见色斑。质地细腻、均匀，触感光滑。容器倾斜时，轻微搅拌下混合物容易流动。轻微团聚。颗粒结构稳定。5 完全均匀，整个样品视觉上一致。完全无结块。质地均匀。颜色强度完全均匀，在整个配方中分散良好。丝滑。无任何可触及的粗糙感。混合物无需任何搅拌即可轻松流动。无团聚，混合物保持分散。图3 用于混合试验中配方成分的混合程序。该程序将称为方法B。含CA-EO的配方类似应用相同程序。CA-EO：Cryptocarya alba精油。LP-EO：Laureliopsis philippiana精油。MgS：硬脂酸镁。SLES：月桂醇聚醚硫酸酯钠。
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science06frontiersin.org，稳定性、机械稳定性及长期稳定性评价（28, 29）。除宏观稳定性评估外，还对最终制剂进行了补充理化表征。采用扫描电子显微镜（SEM）检查颗粒形态和表面特征。进行傅里叶变换红外光谱（FTIR）分析，以比较 SCA、SLP 和 SVE 与 CA-EO 和 LP-EO 的光谱特征，同时采用差示扫描量热法（DSC）评估制剂和分离精油的热行为。通过加热后质量损失以重量法测定水分含量。通过松装体积和振实体积测量评价粉末流动性，并据此计算豪斯纳比（HR）和卡尔指数（CI）。HR 计算为振实密度与松装密度之比，而 CI 计算为振实密度与松装密度之间的相对差值。每种制剂的 HR 和 CI 测量均进行三次重复，而水分含量为每种制剂单次测定。详细的 SEM、FTIR、DSC、水分、HR 和 CI 结果见补充材料。pH 采用以 1:10 比例（香波:蒸馏水）制备的复水溶液测定，将 0.5 g 样品与 4.5 mL 蒸馏水混合。使用先前校准的 AQUASEARCHER™ AB23PH pH 计进行测量，以评估制剂相容性并检测与不稳定或降解过程相关的潜在变化（28, 29）。由于制剂开发为干粉原型，在生物学评价前未进行 pH 调节。调整所选粉末系统的 pH 将需要重新配制并随后重新评估均匀性、粉末流动性、精油分布和物理稳定性。因此，所选原型在未进一步调节 pH 的情况下进行评价，而碱性 pH 被视为制剂局限性，将在后续优化阶段解决。热稳定性通过将各制剂等分试样在 50 °C 孵育 120 h 进行评估，每 24 h 进行感官评价，而长期稳定性通过室温台面储存 9 个月进行评估。在两项测试中，均评价表 1 中所述的感官参数。机械稳定性通过离心作为加速物理稳定性测试进行评估，因为离心应力可促进颗粒重排并揭示在静态储存条件下可能不明显的压实、相分离、沉降或粉末均匀性丧失倾向（29, 31）。简言之，将 0.2 g 等分试样在室温下以 13,000 rpm 离心 30 min，以检测压实、絮凝或沉降迹象。这些结果采用表 2 中所述的定性标准进行评估（28, 29, 31）。热、机械和长期试验主要设计用于评估宏观和物理稳定性。未对储存期间精油进行定量纵向化学谱分析。2.5 最终制剂的体外抗菌评价 2.5.1 琼脂扩散试验 最终干香波制剂对假中间葡萄球菌 ATCC 49444 和三株临床分离株（SP 1、SP 9 和 SP 13）的抗菌活性进行了评价。采用琼脂扩散试验评估 SCA、SLP 和 SVE，并以 2% 氯己定作为参考消毒剂对照，未处理培养物作为生长对照。孵育后以毫米为单位测量抑菌圈直径，试验进行三次重复。完整结果见补充材料。2.5.2 倍比稀释和最低抑菌浓度（MIC）试验 最终制剂还采用倍比稀释试验进行评价。测试前，将干香波以 1:3 比例分散于 Mueller–Hinton 肉汤中。SCA 和 SLP 含有各自精油的 3%，因此稀释系列以等效精油浓度表示。评价浓度范围为 30.72 至 0.06 μg/mL。SVE 不含精油，按相应制剂稀释水平评价，并作为载体对照。每次试验均包括阳性和阴性生长对照。最低抑菌浓度（MIC）确定为在所评价条件下与无可检测细菌生长相关的最低测试等效精油浓度。完整稀释数据见补充材料。2.5.3 活菌计数 为提供细菌存活的定量评估，将倍比稀释试验的等分试样进行培养，并计算活菌数。表 2 机械稳定性测试评价标准。评分 压实 絮凝 沉降 1 粉末完全压实；形成固体且难以处理的一团 形成大絮体或聚集体；明显可见的团块不分散 粉末分离为明显层次；观察到高度沉降 2 粉末可见压实；团块经搅动后可分离 轻微絮凝；存在分散聚集体；团块经搅动后分散 中度沉降，部分颗粒仍悬浮 3 未观察到压实；粉末保持松散 未观察到絮凝；混合物无可见聚集体 未观察到沉降；颗粒保持完全悬浮
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science07frontiersin.org，恢复情况以每毫升菌落形成单位（CFU/mL）定量。CFU mL⁻¹ 值按计数菌落数×1,000 计算。这些测定针对假中间葡萄球菌 ATCC 49444 及临床分离株 SP 1、SP 9 和 SP 13 进行。各制剂及各稀释度的完整 CFU/mL 结果见补充材料。2.6 临床前安全性试验 在犬进行探索性微生物学与耐受性评估之前，先在小鼠中进行了临床前安全性试验，包括皮肤刺激性测量和离体组织学分析。为此，于2025年4月在智利大学牙学院实验平台获取了18只健康雌雄 BALB/c 小鼠（Mus musculus），以涵盖生物学变异。动物分为三组，每组6只。根据所用制剂，分组如下：C. alba 精油负载干香波治疗组（SCA）、L. philippiana 精油负载干香波治疗组（SLP）和赋形剂治疗组（SVE）。所有动物均按照智利大学牙学院伦理委员会（CICUA：24814-ODO-UCH-e3）的规定进行饲养。后续在犬中进行的疗效试验仅在获得主人书面知情同意及相应动物伦理委员会批准后进行。所有小鼠均饲养于智利大学牙学院的常规设施内，房间温度可控，光照周期为12小时明:12小时暗。6月9日，对所有动物称重，并使用电动剪毛器小心去除背部1×2 cm区域的毛发，不使用化学脱毛剂。首次制剂涂抹于去毛后1小时进行。在产品涂抹后1、4和8小时记录刺激、炎症和不良体征。8小时观察期结束后给予第二剂制剂。在刺激性试验的第2、3和4天，执行相同的涂抹方案。在刺激性试验第5天，所有动物通过受控CO₂暴露实施安乐死。随后立即采集背部处理皮肤的皮肤样本，转移至含10%甲醛的15 mL离心管中用于组织学分析（18, 20, 22）。2.6.1 皮肤刺激性测量 进行皮肤刺激性试验以评估与香波涂抹相关的皮肤刺激程度。采用两项标准描述刺激性，详见表3。2.6.2 离体组织学分析 对于组织学评估，首先将5 μm组织切片用二甲苯（Biopack）脱蜡15分钟。随后通过递减浓度乙醇（100%、95%、80%、70%和50%）序列水化，每步5分钟。切片用苏木精-伊红（H&E，Biopack）染色5分钟，然后在流水下冲洗2分钟去除多余染液。随后使用递增浓度乙醇（50%、70%、80%、95%和100%）脱水，并用显微镜封片剂（Biopack）封片。干燥后，每组取五个代表性显微镜视野，使用Olympus显微镜在明场下检查。图像使用DFC 295数码相机（Olympus）采集，使用4×物镜和10×目镜，总放大倍数为40×。组织学切片由两名对实验分组不知情的专业病理学家独立评估。根据实验性皮肤损伤模型已确立的特征性组织病理学特征评估组织切片，包括上皮完整性、表皮厚度、上皮细胞密度、炎性细胞浸润、水疱形成、真皮结构、水肿、血管充血及与组织损伤和修复相关的其他形态学改变（32–34）。组织病理学发现根据这些既定标准进行判读。在判读不一致的情况下，共同复审切片直至达成共识，然后确定最终组织病理学分类。组织病理学评估为定性评估，未对评估参数预先设定半定量数值评分。2.7 犬的微生物学评估 2.7.1 研究设计、伦理批准、随机化、分配隐藏和盲法 在临床诊断为浅表性细菌性脓皮病的客户所有犬中开展了一项探索性、前瞻性、随机、对照、四臂平行组临床研究。犬从智利圣地亚哥多家兽医诊疗中心招募，包括私人诊所和大学附属兽医服务机构。研究方案经相应动物伦理委员会批准，批准代码为ID14/2024，日期为2024年6月28日。入组前获得所有主人的书面知情同意。表3 用于评估刺激性的评分系统。评分 红斑 水肿 0 无病变 无水肿 1 轻度发红 轻度水肿 2 中度发红 中等大小水肿（隆起>1 mm）3 严重发红或灼伤 隆起>1 mm并超出处理区域
Varas等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，08frontiersin.org。三十二只犬按1:1:1:1的比例分配至四个平行治疗组，每组计划八只犬：含Cryptocarya alba精油的香波（SCA）、含Laureliopsis philippiana精油的香波（SLP）、赋形剂香波（SVE），或以氯己定为基底的市售对照品Inveclor®。研究期间，临床需要时允许使用奥拉替尼或相当的非皮质类固醇止痒治疗。相反，不允许使用皮质类固醇、全身或局部抗生素、抗菌剂以及具有潜在抗菌或抗炎活性的天然产品。如果在基线评估前15天内接受过全身或局部抗生素、任何类别的皮质类固醇或具有潜在抗菌或抗炎活性的天然产品，犬只不符合入组资格。因此，所有入组犬在微生物采样和开始指定局部治疗前均完成了至少15天的无治疗期。随机分配序列使用Datacapt Randomization平台生成。在个体犬水平上应用简单随机化，无分层、区组或其他限制。随机化序列以及研究代码与治疗组之间的对应关系仅对Daniela Siel可及，其担任分配保管人，未参与盲法临床结局评估、临床实验室分析或微生物学评价。通过将随机化序列和治疗代码清单的访问权限限制于分配保管人来维持分配隐藏。在获取相应治疗分配之前，确认资格、获得书面主人知情同意，并将每只犬入组并分配个别研究识别代码。负责招募和临床评估的临床医生无法访问分配序列，也无法预测下一只入组犬所分配的治疗。所有研究产品均以相同容器分发，容器仅标注相应的研究识别代码。容器未显示制剂名称、精油、赋形剂或对照品身份。尽管所有产品均以相同的编码容器提供，但未正式评估盲法有效性，不能完全排除基于潜在感官差异的治疗识别。犬主人、治疗临床医生以及负责随访和结局评估的临床医生在整个研究期间对治疗分配均处于盲态。临床记录和生物样本仅使用编码研究识别号进行标识。进行血液学和生化分析、细胞学评估、细菌培养、菌落计数评估以及使用VITEK®系统进行细菌鉴定的人员也对治疗分配处于盲态。在完成相应评估和分析之前，治疗代码未向临床或实验室评估人员披露。随机化、分配隐藏、实施和盲法程序的报告按照PetSORT针对客户拥有犬和猫的随机试验建议进行构建（35）。犬研究的主要探索性结局是第21天的微生物学反应，通过细菌培养、细菌检测以及基于CFU计数的预定义细菌负荷类别进行评估。随访期间进行的临床观察被视为局部耐受性和总体临床演变的支持性评估，而非独立的定量疗效结局。由于未前瞻性应用标准化的定量病变严重程度评分，且并非所有入组犬均有完整的标准化照片记录，因此未生成回顾性临床病变评分。分配后，由主人将指定的局部产品直接施用于受影响皮肤区域，每日两次，理想情况下约12小时间隔，连续21天。产品使用防护手套施用，并留在皮肤上不冲洗。每次施用后，指导主人在约10–20分钟内防止犬舔舐、梳理或以其他方式去除产品。向主人提供标准化说明以促进产品施用的一致性。临床随访在第0、7、14和21天进行。2.7.2 微生物采样和细菌鉴定。在治疗开始前和完成21天局部治疗期后，从临床诊断为浅表性细菌性脓皮病的犬采集微生物学标本。根据犬浅表性脓皮病微生物学评价的标准方法（17, 36），使用无菌棉拭子从活动性皮肤病变处取样。采样前，用无菌纱布小心去除病变区域的表面碎屑，不使用抗菌剂，以避免影响细菌回收。将拭子在病变表面轻轻滚动以收集渗出物和细菌物质，样品在冷藏条件下迅速运送至智利大学兽医与动物科学学院MICROVET诊断实验室进行进一步处理。到达后，将样品接种于血琼脂平板，并在37°C下孵育24–48小时。选择形态特征提示葡萄球菌的菌落并传代培养以获得纯细菌分离株。随后使用VITEK®自动鉴定系统（bioMérieux）进行物种鉴定，能够在物种水平鉴定假中间葡萄球菌（19）。对每个样品，记录假中间葡萄球菌的存在或缺失。还考虑检测其他葡萄球菌属以进一步描述病变的细菌谱。分离株分类为假中间葡萄球菌、金黄色葡萄球菌或其他葡萄球菌物种。使用相应的自动药敏检测板评估抗菌药物敏感性。以苯唑西林敏感性作为鉴定耐甲氧西林假中间葡萄球菌（MRSP）的表型指标，对苯唑西林显示耐药的分离株被认为表型上与MRSP相符。为评估对治疗的微生物学反应，通过计数基线和局部干香波施用21天后从病变拭子培养物中回收的菌落形成单位（CFU）来估计细菌负荷。当细菌计数超过300 CFU/拭子时，基线培养物被认为阳性，对应于非常高或不可计数的细菌负荷。治疗后，当检测到0–15 CFU/拭子时，细菌生长分类为缺失或无显著意义；当值为16至
Varas等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，09frontiersin.org，300 CFU/拭子，当细菌计数超过300 CFU/拭子时不可计数。2.8 样本量计算 使用G*Power 3.1.9.6版进行先验样本量计算，并在RStudio 2024.12.1版中通过R 4.4.3版独立验证。预期效应量根据本课题组产生的未发表初步数据估算。对于四个等量分配治疗组之间微生物学反应的总体比较，计算考虑Cohen's w = 0.60、双侧I类错误率0.05和80%统计功效。分析表明，所需最小总样本量为32只犬，相当于每组8只动物。该样本量也与适用于经皮给药兽药产品靶动物安全性和局部耐受性评价的VICH GL43建议一致。本研究设计用于检测较大的微生物学效应和常见耐受性问题，其功效不足以检测小或中等效应，也不足以确定治疗之间的等效性或非劣效性。2.9 统计分析 使用GraphPad Prism 10版和RStudio 2024.12.1版中的R 4.4.3版进行统计分析。统计学方法和修订分析经与生物统计学家协商审查。个体动物被视为实验单位。所有统计检验均为双侧，统计显著性设定为p < 0.05。pH测量为技术重复，因此以均值±标准差进行描述性总结，不进行推断性统计比较。对于使用参数方法分析的连续结局，主要通过残差图形检查评估模型假设，包括分位数-分位数图和残差-拟合值图。模型残差的正态性另外使用Shapiro-Wilk检验评估，方差齐性使用Brown-Forsythe检验评估。由于正式正态性检验在小样本中功效有限，统计方法的选择基于图形诊断、残差分布和方差齐性的综合评估，而非仅基于Shapiro-Wilk检验。当参数假设未充分满足时，使用适当的非参数或稳健替代方法。微生物学结局根据其尺度和数据结构的进行分析。第21天的细菌检测被视为二分类结局，而细菌负荷根据预定义的CFU类别进行分析。由于样本量有限且单元格计数稀疏，第21天的总体组间比较使用Fisher-Freeman-Halton精确检验。计算Cramér's V作为治疗组与分类微生物学结局之间总体关联的效应量指标。细菌检测从基线至第21天的变化使用精确McNemar检验评估，考虑同一犬内重复观察的配对性质。仅纳入在两个时间点均有微生物学结果的动物进行配对分析。细菌物种鉴定的变化使用个体转换频率进行描述性总结，因为若干类别包含稀疏或零单元格计数，无法进行可靠的多分类配对推断。缺失观察未进行插补，并报告每项分析的动物数量。在进行次要成对比较时，使用Holm程序调整p值。对于多项血液学和生化安全性结局，使用Benjamini-Hochberg程序控制错误发现率。在相关情况下报告未调整和调整后的p值，当适用多重性校正时，统计推断基于调整值。缺失观察未进行插补，并报告每项分析的动物数量。3 结果 3.1 均匀性试验 感官参数——外观、无结块、颜色一致性、触感质地、流动性、结块倾向和香气强度（表1）——用于评估制剂均匀性和操作性能，这些是局部用粉末性能和可接受性的关键因素。如表4所示，在玉米淀粉和木薯淀粉基质中，含MgS的制剂比不含MgS的对应制剂获得更高的总体平均分。总体平均分从SLP1的2.6增至SLP1-MgS的4.0，从SLP2的3.4增至SLP2-MgS的4.7。混合淀粉 表4 均匀性试验中评估的所有制剂的感官评分。参数 SLP1 SLP1-MgS SLP2 SLP2-MgS SLP3 SLP3-MgS 视觉均匀性 2.0 4.0 3.0 5.0 2.0 4.0 无结块 2.0 3.0 3.0 4.0 2.0 4.0 颜色均匀性 2.0 5.0 3.0 5.0 3.0 5.0 触感质地 2.0 3.0 4.0 5.0 3.0 4.0 流动性 3.0 4.0 3.0 4.0 3.0 4.0 结块倾向 2.0 4.0 3.0 5.0 3.0 4.0 香气强度 5.0 5.0 5.0 5.0 5.0 5.0 总体平均 2.6 4.0 3.4 4.7 3.0 4.3 SLP，含Laureliopsis philippiana精油的香波。
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，10frontiersin.org。该制剂在加入 MgS 后总体平均评分也有所提高，从 SLP3 的 3.0 升至 SLP3-MgS 的 4.3。在所有评估的制剂中，SLP2-MgS 的总体表现最高，总体平均评分为 4.7，其次是 SLP3-MgS 和 SLP1-MgS，平均评分分别为 4.3 和 4.0。所有制剂的香气强度保持不变，评分为 5。总体而言，含 MgS 的木薯基制剂平均评分最高（4.7），而 SLP1、SLP1-MgS、SLP2、SLP3 和 SLP3-MgS 分别为 2.6、4.0、3.4、3.0 和 4.3。含 MgS 的制剂还表现出较低的团聚倾向和改善的 EO 在基质中的分散性。3.2 混合试验 混合试验结果汇总于表 5。总体而言，方法 A 制备的混合物（SLP1-MA、SLP2-MA 和 SLP3-MA）流动性改善、颗粒内聚力较低，而方法 B（SLP1-MB、SLP2-MB 和 SLP3-MB）产生细小块状物和较大团聚，表明固体基质和 EO 的分散效率较低。颜色均匀性在不同制剂和混合方法之间无差异，表明两种方法在制剂中产生的 EO 分布相同。SLP3-MgS 的块状物形成倾向评分低于 SLP1-MgS 和 SLP2-MgS 制剂，且与所用混合方法无关。3.3 优化制剂的宏观理化表征 3.3.1 pH 测定 pH 测定（表 6）显示 SLP、SCA 和 SVE 之间略有差异。所有三种制剂均表现出相似的碱性值，范围为 10.93 至 11.16，变异性低，这反映在较小的标准差上。这些结果表明，加入精油并未明显改变粉末基质的 pH。鉴于制剂的碱性性质，其用于反复局部给药的适用性以及对皮肤耐受性的潜在影响将在第 4.2.1 节进一步讨论。3.3.2 热稳定性 热稳定性结果（表 7–9）显示，随着孵育时间增加，感官和物理性质逐渐变化。在 120 小时测定期间，视觉均匀性和颜色均匀性保持不变，而触觉质地、流动性、表 5 在混合试验中使用混合方法 A 和方法 B 评估的制剂获得的感官评分。参数 SLP1-MA SLP1-MB SLP2-MA SLP2-MB SLP3-MA SLP3-MB 视觉均匀性 5.0 4.0 4.0 3.0 4.0 3.0 无块状物 4.0 3.0 3.0 3.0 3.0 3.0 颜色均匀性 5.0 5.0 5.0 5.0 5.0 5.0 触觉质地 4.0 4.0 4.0 3.0 4.0 4.0 流动性 5.0 5.0 4.0 4.0 3.0 2.0 团聚倾向 4.0 3.0 4.0 3.0 2.0 2.0 香气强度 3.0 3.0 3.0 3.0 3.0 3.0 总体平均值 4.3 3.9 3.9 3.4 3.4 3.1 SLP，含 Laureliopsis philippiana 精油的洗发剂，通过方法 A 和方法 B（MA、MB）制备。表 6 所有制剂的 pH 值。制剂 pH SLP 10.93 ± 0.06 SCA 11.01 ± 0.03 SVE 11.16 ± 0.03 SLP，含 Laureliopsis philippiana 精油的洗发剂；SCA，含 Cryptocarya alba 精油的洗发剂；SVE，赋形剂洗发剂。表 7 载有 Laureliopsis philippiana 精油（SLP）的洗发剂的热稳定性评分。参数 0 24 48 72 96 120 视觉均匀性 4.0 4.0 4.0 4.0 4.0 4.0 无块状物 3.5 3.0 3.0 3.0 3.0 3.0 颜色均匀性 4.0 4.0 4.0 4.0 4.0 4.0 触觉质地 4.0 3.0 3.0 2.0 2.0 2.0 流动性 4.0 4.0 4.0 3.5 3.0 3.0 团聚倾向 3.5 3.0 3.0 3.0 2.0 2.0 香气强度 4.0 2.0 2.0 2.0 2.0 2.0 总体平均值 3.9 3.3 3.3 3.1 2.9 2.9 表 8 载有 Cryptocarya alba 精油（SCA）的洗发剂的热稳定性评分。参数 0 24 48 72 96 120 视觉均匀性 4.0 4.0 4.0 4.0 4.0 4.0 无块状物 3.5 3.0 2.5 2.5 2.0 3.0 颜色均匀性 4.0 4.0 4.0 4.0 4.0 4.0 触觉质地 4.0 4.0 3.0 2.5 2.5 2.5 流动性 4.0 4.0 4.0 3.7 3.0 3.0 团聚倾向 3.5 3.0 3.0 3.0 2.0 2.0 香气强度 3.0 2.5 2.0 2.0 2.0 2.0 总体平均值 3.8 3.5 3.2 3.0 2.8 2.8
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，11，frontiersin.org。结块倾向和香气强度评分呈进行性下降。两种含精油制剂在热暴露期间香气强度均降低，与挥发性成分损失一致；然而，本试验期间未对精油的化学成分进行定量评估。因此，这些结果被解释为在所评估热条件下初步物理稳定性的证据，而非化学稳定性的确认。3.3.3 机械稳定性。离心数据（表10）显示，所有制剂均获得2分的压实评分。对于絮凝，SLP和SCA获得2分，而SVE得分最高，为3分。对于沉降，SLP和SVE获得3分，而SCA得分较低，为2分。总平均分SLP为2.3分，SCA为2分，SVE为2.7分，SVE在机械稳定性试验中总平均分最高，其次是SLP和SCA。3.3.4 长期稳定性。长期稳定性试验结果（表11–13）显示，在9个月的评估期内，所测试的三种制剂（SLP、SCA和SVE）的若干宏观性质得以保持。在所有制剂中，视觉均匀性和颜色均匀性在整个9个月评估期内保持不变。对于SLP，总平均分从基线的3.9分降至3个月时的3.0分，并在6个月和9个月时均为2.9分。对于SCA，总平均分从基线的3.7分降至3个月和6个月时的2.9分，并在9个月时降至2.7分。对于SVE，总平均分从基线的4.0分降至3个月时的3.6分，并在6个月和9个月时均为3.4分。在两种含精油制剂中，香气强度在3个月后从5分降至3分，并保持该评分直至第9个月，而SVE在整个试验期间保持无气味。与触觉质地、流动性和结块倾向相关的评分随时间推移而降低，而无结块在SVE中保持不变，在两种含精油制剂中于3个月后下降。总体而言，在九个月储存期内，视觉均匀性和颜色均匀性得以维持，而在质地、流动性、结块倾向和香气强度方面观察到逐渐变化。这些结果表明制剂的宏观外观得以保持，而粉末处理性质随时间推移发生进行性变化。由于未对精油成分进行定量化学分析，表9 SVE洗发剂的热稳定性评分。参数 0 24 48 72 96 120。视觉均匀性 4.0 4.0 4.0 4.0 4.0 4.0。无结块 4.0 3.0 3.0 3.0 2.5 2.0。颜色均匀性 5.0 5.0 5.0 5.0 5.0 5.0。触觉质地 4.0 4.0 4.0 3.5 3.0 2.5。流动性 4.0 4.0 4.0 4.0 3.5 3.0。结块倾向 3.0 3.0 3.0 3.0 2.5 2.5。香气强度 0.0 0.0 0.0 0.0 0.0 0.0。总平均 3.4 3.3 3.3 3.2 2.9 2.7。表10 负载SLP、SCA和SVE的洗发剂评分。参数 SLP SCA SVE。压实 2.0 2.0 2.0。絮凝 2.0 2.0 3.0。沉降 3.0 2.0 3.0。总平均 2.3 2.0 2.7。表11 负载Laureliopsis philippiana精油（SLP）洗发剂的长期稳定性评分。参数 0个月 3个月 6个月 9个月。视觉均匀性 4.0 4.0 4.0 4.0。无结块 3.0 2.0 2.0 2.0。颜色均匀性 4.0 4.0 4.0 4.0。触觉质地 3.0 2.0 2.0 2.0。流动性 4.0 3.0 2.0 2.0。结块倾向 4.0 3.0 3.0 3.0。香气强度 5.0 3.0 3.0 3.0。总平均 3.9 3.0 2.9 2.9。表12 SCA评分。参数 0个月 3个月 6个月 9个月。视觉均匀性 4.0 4.0 4.0 4.0。无结块 3.0 2.0 2.0 2.0。颜色均匀性 4.0 4.0 4.0 4.0。触觉质地 3.0 2.0 2.0 2.0。流动性 4.0 3.0 3.0 2.0。结块倾向 3.0 2.0 2.0 2.0。香气强度 5.0 3.0 3.0 3.0。总平均 3.7 2.9 2.9 2.7。表13 SVE评分。参数 0个月 3个月 6个月 9个月。视觉均匀性 4.0 4.0 4.0 4.0。无结块 4.0 4.0 4.0 4.0。颜色均匀性 5.0 5.0 5.0 5.0。触觉质地 5.0 4.0 4.0 4.0。流动性 5.0 4.0 4.0 4.0。结块倾向 5.0 4.0 3.0 3.0。香气强度 0.0 0.0 0.0 0.0。总平均 4.0 3.6 3.4 3.4
Varas等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science 12，frontiersin.org。若在贮藏期间进行，这些观察结果应解释为物理稳定性而非化学稳定性的初步证据。3.3.5 补充理化表征 通过扫描电子显微镜（SEM）、傅里叶变换红外光谱（FTIR）、差示扫描量热法（DSC）、水分测定和粉末流动性测量对最终干洗香波制剂进行了补充理化表征（补充图S1–S3和补充表S4）。SEM检查显示SCA、SLP和SVE中颗粒形态不均一，整个样品中可见颗粒结构和颗粒聚集体（补充图S1）。FTIR分析显示SCA、SLP和SVE之间的光谱轮廓大体相似，制剂光谱主要以与常见粉末基质相关的谱带为特征（补充图S2）。分离的CA-EO和LP-EO表现出不同的光谱特征，特别是在指纹区内，而与载体制剂相比，含EO制剂中未观察到主要的新吸收谱带。DSC热分析图显示三种干洗香波制剂在评估温度范围内具有相似的总体热谱（补充图S3）。SCA和SLP显示的热事件与SVE中观察到的相当，而分离的精油在分析范围内显示相对有限的热转变。在配制粉末中未发现明显仅归因于EO掺入的额外热事件。粉末流动性测量显示SCA、SLP和SVE的Hausner比分别为1.62 ± 0.02、1.63 ± 0.08和1.70 ± 0.06。相应的Carr指数值分别为38.30 ± 0.74%、38.39 ± 3.09%和40.92 ± 3.47%（补充表S4）。水分含量为SCA 12.61%、SLP 14.02%和SVE 10.69%。总体而言，SCA和SLP显示相似的流动相关参数，而SVE呈现最高的Hausner比和Carr指数。3.4 初步抗菌活性 作为干洗香波制剂抗菌活性的初步评估，针对选定的假中间葡萄球菌临床分离株进行了琼脂扩散试验（补充表S5）。SCA和SLP对三种测试临床分离株均未产生可测量的抑菌圈，而2%氯己定对照显示明显抑制，平均抑菌圈范围为19.73至23.85 mm。这些发现表明干洗香波制剂在琼脂扩散试验条件下未表现出可检测的抗菌活性。作为补充探索性评估，通过肉汤微量稀释法针对选定的假中间葡萄球菌菌株评估了干洗香波制剂的抗菌活性（补充表S6），并进行了相应稀释水平的定量活菌计数（补充表S7–S10）。与琼脂扩散结果相反，SCA和SLP在等效EO浓度0.12至0.24 μg/mL范围内对测试菌株显示抑制活性。活菌计数数据显示较高制剂浓度下细菌回收率较低，随着制剂逐步稀释CFU/mL增加（补充表S7–S10）。然而，载体制剂（SVE）也观察到抑制反应和细菌回收率降低，表明观察到的反应不限于含EO制剂。3.5 刺激性结果 所有皮肤刺激性测量评分均为0，表明含EO制剂和载体均未在处理部位产生红斑或水肿。连续摄影记录（图4）使得能够评估皮肤 图4 三只小鼠剃毛背部区域的摄影记录（第一排：含LP-EO的原型；第二排：含CA-EO的原型；第三排：安全性试验期间不含EO的载体），显示无明显宏观皮肤改变。
Varas等，10.3389/fvets.2026.1918882，《前沿兽医科学》，frontiersin.org，在剃毛并标记的背侧区域（对应制剂施用部位）出现反应。在整个观察期内，动物皮肤保持完整，无肉眼可见的皮肤刺激征象，包括红斑、水肿、溃疡、渗出、严重脱屑、开放性病变、结痂形成、出血或坏死区域。处理区域随时间推移保持规则边界和均匀色泽。一般福利参数——包括外观、食物和饮水量、体温、呼吸和心率，以及自发和被诱发行为——在整个实验期间均保持在正常范围内。研究期间体重出现轻微波动，包括总体低于5%的增加和减少，在个体、笼位或处理组之间无一致模式。随访期内未观察到进行性或持续性体重减轻。3.6 组织学分析 采集小鼠背侧皮肤组织样本，以在细胞水平评估与制剂施用相关的潜在刺激、炎症或组织损伤征象。组织学分析（图5）显示皮肤结构似保持完好，表皮连续且界限清晰，无溃疡、糜烂或明显增生证据。其下真皮维持正常的胶原纤维排列，无明显炎症浸润或间质水肿征象。同样，可见的皮肤附属器形态保持完好，与健康组织一致。综上所述，比较组织学分析表明，所评估的制剂均未诱导动物皮肤出现明显的组织病理学改变，结果与生理正常组织一致。3.7 犬的微生物学评估 为提供在目标物种中抗菌活性的初步证据，对诊断为浅表性脓皮病的客户所有犬进行了微生物学评估。犬最初被分配至四个治疗组。然而，赋形剂香波组的一只犬在最终微生物学评估前退出研究；因此，最终微生物学分析包括SCA组八只犬、SLP组八只犬、SVE组七只犬和Inveclor ®组八只犬。基线时，除一只犬外，所有犬葡萄球菌属培养均为阳性，其中假中间葡萄球菌为鉴定出的优势菌种。第21天时，假中间葡萄球菌仍为最常检测到的菌种；然而，在一些动物中观察到细菌鉴定结果的变化，包括S. 图5 从小鼠表皮获得的组织学样本，按香波制剂分组。图像通过光学显微镜使用4×物镜和10×目镜获取，对应总放大倍数40×。SLP：含LP-EO的香波，SCA：含CA-EO的香波，SVE：不含EO的赋形剂香波。表14 浅表性脓皮病犬（n = 31）在第1天至第21天之间通过VITEK®鉴定的细菌菌株。第1天鉴定菌株 第21天鉴定菌株 假中间葡萄球菌 金黄色葡萄球菌 其他菌种 无细菌 假中间葡萄球菌 15 5 2 8 金黄色葡萄球菌 0 0 0 0 其他菌种 1 0 0 0 数据表示在基线（第1天）和治疗结束（第21天）之间显示细菌菌种每种转变的犬数。“其他菌种”包括绿色气球菌和沃纳葡萄球菌。“无细菌”表示第21天培养中未检测到球菌。
Varas 等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science 14，frontiersin.org。假中间葡萄球菌和金黄色葡萄球菌、向其他葡萄球菌种的转变，以及培养中未检出球菌的病例（表14）。观察到若干转变，包括假中间葡萄球菌持续存在、向其他葡萄球菌种转变，以及第21天无可检出球菌。由于若干类别细胞计数稀少或为零，未对多分类物种转变进行正式的推断性比较。第21天，在SCA组和Inveclor ® 组实现了细菌消除（定义为培养中无可检出球菌），但在SVE组和SLP组未实现。第21天细菌载量类别的分布在各治疗组间数值上存在差异，但未达到统计学显著性（Fisher–Freeman–Halton精确检验，p = 0.639；Cramér's V = 0.277）。细菌消除率最高的是Inveclor ® 组，8只犬中有4只（50.0%）无可检出球菌。在含精油香波组中，仅在SCA组观察到细菌消除，8只犬中有3只（37.5%）在第21天无可检出球菌。相比之下，SLP组所有犬仍为培养阳性，赋形剂香波组（SVE）所有犬亦如此，分别有8/8和7/7只犬检出细菌（表15）。尽管效应量估计提示治疗组与细菌载量类别之间存在不可忽略的关联，但组样本量小且细胞计数稀少导致相当大的不确定性。因此，这些发现应被解释为探索性结果。此外，第21天对细菌载量进行了分类，以更详细地评估微生物学应答。达到非显著细菌载量类别的犬比例最高的是SCA组（4/8；50.0%），其次是Inveclor ® 组（3/8；37.5%）、SVE组（2/7；28.6%）和SLP组（1/8；12.5%）。可计数细菌载量见于SCA组8只犬中的2只（25.0%）、SLP组8只犬中的1只（12.5%）、SVE组7只犬中的2只（28.6%）和Inveclor ® 组8只犬中的2只（25.0%）。仍处于不可计数类别的犬比例最高的是SLP组（6/8；75.0%），其次是SVE组（3/7；42.9%）、Inveclor ® 组（3/8；37.5%）和SCA组（2/8；25.0%；表16）。使用Fisher–Freeman–Halton精确检验比较各治疗组间细菌载量分布，未发现统计学显著差异（p = 0.8781）。第21天，将细菌生长分类为非显著或显著，以评估各治疗组间的微生物学应答（图6）。非显著细菌生长的犬比例最高的是SCA组，8只犬中有4只（50.0%），其次是Inveclor ® 组，8只犬中有3只（37.5%）。在SVE组中，2只犬观察到非显著生长，5只显示显著生长，1只无可用信息。在SLP组中，1只犬在第21天显示非显著细菌生长，其余病例观察到显著细菌生长。当根据预定义的CFU阈值将细菌生长二分类时，各治疗组间未检测到统计学显著的总体差异（Fisher–Freeman–Halton精确检验（p = 0.485）；Cramér's V = 0.2）。SCA组达到较低细菌生长类别的犬比例最高。然而，置信区间较宽，估计的组间差异仍不精确。抗菌药物敏感性试验显示，研究期间回收的假中间葡萄球菌分离株均不对苯唑西林耐药。因此，未检测到表型上与MRSP相容的分离株。4 讨论 4.1 香波配方 4.1.1 均匀性试验 MgS是药物和化妆品粉末体系中已确立的赋形剂，常用作润滑剂和抗黏附剂，通过减少颗粒间摩擦和凝聚来改善流动性（26, 27, 37）。这些特性被认为与本研究的 表15 浅表性脓皮病犬第21天按治疗组的细菌存在与缺失。组n存在缺失 SLP8 8 0（0.0%）SCA8 5 3（37.5%）SVE7 7 0（0.0%）Inveclor®8 4 4（50%）存在指第21天通过培养和VITEK®鉴定检出任何葡萄球菌种。缺失指培养中无可检出球菌。SCA，含Cryptocarya alba精油香波；SLP，含Laureliopsis philippiana精油香波；SVE，赋形剂香波。表16 第21天按治疗组的细菌载量分类。细菌载量根据第21天CFU计数分类。非显著：0–15 CFU/拭子；可计数：16–300 CFU/拭子；不可计数：>300 CFU/拭子。组n非显著可计数不可计数 SLP8 1（12.5%）1（12.5%）6（75.0%）SCA8 4（50.0%）2（25.0%）2（25.0%）SVE7 2（28.6%）2（28.6%）3（42.9%）Inveclor®8 3（37.5%）2（25.0%）3（37.5%）图6 各治疗组最终微生物培养（第21天）中球菌的显著生长（>15 CFU）或非显著生长。Fisher–Freeman–Halton精确检验未显示各治疗组间存在显著差异（p = 0.485）。灰色表示数据不可用。SLP：含L. philippiana精油香波；SCA：含C. alba精油香波；SVE：不含抗菌药物的赋形剂香波。
Varas等人 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science15frontiersin.org 淀粉基配方，因为掺入亲脂性精油可能会促进团聚、分布不均以及增加粉末基质内的内聚性。与此一致，含MgS的配方（SLP1-MgS、SLP2-MgS和SLP3-MgS）与其不含MgS的对应配方相比，表现出更好的视觉均匀性、更佳的流动性和更低的团聚倾向（表4）。这些特性可能有利于精油在粉末基质内更均匀地掺入，并在局部应用过程中实现更一致的操作（10, 38）。各配方之间观察到的相似芳香强度表明，在初始评价时感官香气强度相当。总体而言，这些发现支持将MgS纳入所选配方，以进行后续的理化表征和稳定性测试（31, 39）。尽管其他助流辅料也可能适用，但本初步优化阶段旨在评估MgS的功能贡献，而非对助流剂或抗结块剂进行全面筛选。4.1.2 混合试验 在含有挥发性亲脂性化合物的粉末系统中，高效混合尤为重要，因为充分的分散有助于配方均匀性以及活性成分在粉末基质内更一致的分布。比较两种混合方法（图2、图3），关键差异之一是淀粉源、精油和MgS的混合顺序。鉴于MgS作为抗团聚剂的作用，混合方法A中MgS与淀粉源的初始混合步骤在视觉均匀性和团聚倾向方面总体表现优于方法B（表5）。两种方法制备的样品之间芳香强度保持相似，表明两种混合方法之间未检测到明显的香气强度感官差异（表5）（38, 39）。综合考虑均匀性和混合评价的结果，选择含MgS且采用方法A制备的玉米淀粉基配方作为后续研究的基础原型。尽管SLP2-MgS在初始均匀性测试中获得了最高的单项平均分，但玉米淀粉基配方在视觉均匀性、流动行为、团聚倾向以及所选混合程序下的性能方面表现出最有利的总体平衡。另一个局限性涉及配方优化过程的初步性质。感官参数由单一非盲评价者评估，每种配方在每个相应时间点仅评估一次；因此，未进行独立重复评估以及观察者间或观察者内一致性分析，可能在选择优化配方时引入观察者依赖性偏倚。此外，每个最终原型仅制备为单一实验批次，无法评估批间重现性。尽管本研究纳入的补充定量和分析表征，包括水分含量、Hausner比、Carr指数、SEM、FTIR和DSC分析，为所选配方的工艺和理化性质提供了额外的客观信息（29, 31），但这些测量并不能消除与初始定性筛选过程或缺乏独立批次重复相关的局限性。因此，未来的配方开发研究应纳入重复定量测量、多名评价者的盲法评估、配方选择早期阶段的客观粉末表征参数以及独立制备的批次，以确认理化特性、粉末流动行为、精油分布和稳定性在各生产批次间的重现性。4.2 理化表征与稳定性 4.2.1 pH 所获得的碱性值与用于干洗的粉末基配方的性质一致，其中吸收性辅料的存在可能产生微碱性环境。在掺入精油的固体基质中也描述了类似行为（10, 38）。SLP、SCA和SVE观察到的pH值相当，表明精油的掺入未明显改变该参数。然而，所有配方均表现出接近11的碱性pH值，高于犬皮肤最常报道的范围。犬皮肤pH值通常报道在5.5至7.2之间，尽管也有描述更宽的范围为4.84至9.95（40）。此外，市售非药用犬用香波据报道pH值范围为5.7 ± 0.09至8.4 ± 0.18（41）。这一差异具有相关性，因为长时间暴露于高碱性条件可能影响表皮屏障功能，并可能增加对刺激的易感性，尤其是在发炎或受损的皮肤中。在本研究中，pH是在干粉以1:10香波:水比例复水后测量的，因此不一定代表干配方局部应用后产生的确切微环境。尽管如此，碱性值仍是当前原型的重要局限性，特别是考虑到所报道的犬皮肤生理pH范围以及市售犬用香波的pH值（40, 41）。在生物学研究之前未进行pH调整，因为所选配方已作为干粉系统进行了优化，修改该参数将需要重新配制，随后重新评估均匀性、粉末流动性、精油分布和物理稳定性（28, 29, 31）。尽管在评估条件下的小鼠安全性试验中未观察到红斑、水肿或组织学改变，但这些发现不能排除与重复或长时间应用于犬皮肤相关的可能影响。这一考虑具有相关性，因为局部耐受性可能取决于暴露条件和皮肤屏障状态，尤其是在受损或发炎的皮肤中（18, 40）。因此，未来的配方开发应包括pH优化，随后在目标物种中重新进行理化表征和安全性评估，之后才能扩展临床使用。4.2.2 热稳定性试验 热稳定性结果（表7–9）显示，随着孵育时间增加，感官和物理性质逐渐变化。均匀性和颜色等视觉属性在整个试验期间保持不变，表明配方在评估的热条件下宏观外观得以保持。两种含精油配方（SLP和SCA）表现出相似的行为，粉末流动性和触感质地随时间略有下降，并伴有
Varas 等，10.3389/fvets.2026.1918882，《 Frontiers in Veterinary Science》，16，frontiersin.org。聚集倾向逐渐增加且香气强度降低，这与挥发性精油成分的部分损失相符。加入制剂中的精油含有多种挥发性成分。如先前所表征，LP-EO 主要由芳樟醇、桉叶油素和黄樟脑组成，而 CA-EO 显示出更复杂的特征，其中桧烯和桉叶油素为其主要成分之一（23）。这些化合物的挥发性可能有助于在暴露于高温期间香气强度的变化（38）。视觉参数的保持表明在热暴露期间未发生重大宏观不稳定性，而质地、聚集倾向和流动性的渐进性变化表明粉末的物理行为随时间发生了改变。在热孵育期间，SVE 的流动性和无结块评分逐渐降低，表明粉末处理特性随时间发生了变化。总体而言，结果表明含精油的制剂保持了与赋形剂大致相当的宏观稳定性，主要变化仅限于在高温条件下质地和香气的逐渐变化（31, 38）。重要的是，热试验主要评估了宏观和物理特性的变化。尽管香气强度的降低与挥发性成分的损失相符，但在热暴露期间未对精油的化学组成进行定量监测。精油是复杂的混合物，其成分可能根据温度和储存条件发生挥发、氧化和化学降解（38）。因此，不能排除单个精油成分的氧化、降解或选择性挥发，目前的数据不应被解释为证明了完全的化学稳定性。需要进行定量纵向分析，例如在热暴露前后进行 GC-MS 谱分析，以确认单个精油成分的稳定性（31）。4.2.3 离心稳定性试验 离心稳定性试验（表10）显示，所有制剂在离心后均呈现相同程度的压实。在絮凝和沉降方面观察到差异：SVE 未显示可见絮凝，而 SLP 和 SCA 表现出轻微絮凝；此外，仅在 SCA 中观察到中度沉降。尽管这些发现表明制剂之间的机械行为存在一些差异，但未观察到完全压实、大聚集体或明显的相分离，表明粉末系统在评估的离心应力下保持了其整体宏观完整性（29）。总体而言，结果支持所开发粉末原型在评估的离心条件下的初步机械稳定性。4.2.4 长期稳定性试验 在室温下进行9个月的长期稳定性试验显示，SLP、SCA 和 SVE 制剂的若干宏观和物理特性得以保持（表11-13）。颜色和总体均匀性等视觉属性在整个研究期间保持稳定，表明制剂的整体宏观外观得以保持。约3个月后观察到质地、粉末流动性和无结块评分略有下降，这可能反映了储存期间颗粒间相互作用和粉末内聚力的渐进性变化（29）。在含精油的制剂中，香气强度随时间降低，这与挥发性成分的部分损失相符，尽管特征性香气仍可检测到。此类变化是合理的，因为精油成分可能在储存期间根据环境和制剂条件发生挥发、氧化或降解（38）。总体而言，观察到的变化是渐进的，主要与粉末处理特性和香气相关，而非制剂均匀性的可见损失。长期观察与加速热观察之间的一致性进一步支持了以下解释：原型在评估条件下保持了初步的物理稳定性（29, 31）。结合 pH 和离心评估，这些发现为制剂在评估条件下的物理行为提供了补充证据，尽管随时间观察到质地、流动行为和香气的中度变化。本研究的另一个局限性是未在储存期间定量评估精油成分的化学稳定性。尽管制剂在评估条件下保持了其主要物理和感官特性，但这些观察结果不能确定所加入精油的化学组成得以保持。精油成分，特别是挥发性化合物，可能在储存期间受到挥发、氧化或化学降解的影响（38）。因此，未来的研究应包括在基线和规定储存期后对代表性挥发性成分进行定量 GC-MS 谱分析，以表征组成变化并更准确地确定制剂保质期（31）。4.2.5 补充物理化学表征 补充物理化学分析为所选干洗发水原型的技术特性提供了额外的客观信息。SEM 检查显示 SCA、SLP 和 SVE 中颗粒形态不均一，在各制剂中观察到颗粒结构和颗粒聚集体。这些发现与多组分粉末系统的不均一性质一致，其中颗粒形态、表面特性和颗粒间相互作用的差异可能有助于内聚力和粉末流动行为（26, 28, 42）。然而，SEM 观察是定性的，不应被解释为颗粒尺寸分布的定量评估或单个制剂成分分布的直接证据。所有三种制剂获得的 Hausner 比和 Carr 指数值与内聚性粉末行为和有限的内在流动性一致。SCA 和 SLP 显示相似的值，而 SVE 呈现最高的 HR 和 CI 值。这些客观测量补充了制剂开发期间获得的定性观察，并表明最终制剂仍为内聚性粉末系统，可能需要进一步优化以实现可重复的处理和潜在的放大生产（26, 27, 29）。水分含量范围为10.69%至14.02%；然而，由于每种制剂的水分含量仅测定一次，这些值应被描述性地解释，而非作为制剂间差异的证据。水分也与粉末相关
Varas等，10.3389/fvets.2026.1918882，《Frontiers in Veterinary Science》17 frontiersin.org，因为其在储存过程中可改变颗粒间相互作用、团聚和流动行为。FTIR分析显示，SCA、SLP和SVE的光谱轮廓总体相似，成品制剂的光谱主要以与普通粉末基质共有的特征为主。掺入EO后未出现主要的新吸收带，提示未检测到制剂整体红外轮廓的明显变化。然而，这一观察并不能排除EO与赋形剂之间更细微的理化相互作用，仅凭FTIR无法确定化学相容性或稳定性(43)。DSC热分析图同样显示，SCA、SLP和SVE的总体热特征相当，未出现可完全归因于EO掺入的明显额外热事件。这些发现提示，在所评价的温度范围内，掺入CA-EO或LP-EO未使粉末基质的热行为产生可检测的重大变化。然而，缺乏明显的EO相关热事件不应被解释为挥发性EO成分在化学上保持不变的证据。FTIR和DSC可提供制剂系统内光谱特征和热行为的补充信息(43)，但不能替代对化学稳定性的直接定量评估。因此，仍需进行纵向化学谱分析，如GC-MS，以确定EO成分在储存期间的组成稳定性(31, 38)。本研究未直接测定粒径分布和休止角。特别是，SEM分析提供了关于颗粒形态和聚集的定性信息，但不应被解释为粒径分布的定量测定。未来的制剂开发和放大研究应纳入这些额外的粉末表征参数，以更全面地评估流动行为和制造重现性。4.3 最终制剂的体外抗菌活性对成品干洗发香波制剂的直接评价显示，其抗菌行为依赖于检测方法。在琼脂扩散试验中，SCA和SLP对假中间葡萄球菌ATCC 49444或所评价的三株临床分离株均未产生可测量的抑菌圈，而2%氯己定对照产生的抑菌圈约为20–24 mm。这与先前报道的分离CA-EO和LP-EO对假中间葡萄球菌的活性形成对比(23)，表明对分离EO所证明的抗菌活性不能必然外推至成品制剂。掺入固体基质可能影响亲脂性EO成分的可利用性、释放和扩散，而琼脂扩散本身在很大程度上取决于抗菌化合物在琼脂介质中迁移的能力(10, 38, 44)。因此，无可测量抑菌圈应在制剂和检测方法的背景下解释，而不应作为完全无抗菌活性的确定性证据。相比之下，系列稀释实验显示，随着制剂浓度降低，活菌回收率存在差异。SCA和SLP在较高测试浓度下使可检测细菌生长消失或显著减少，随后在更高稀释度下CFU/mL增加，支持液相条件下的浓度依赖性反应。基于稀释的药敏试验使微生物与受试抗菌系统之间接触更直接，因此可补充基于扩散的方法(45)。重要的是，SVE在若干相应制剂稀释水平下也降低了细菌回收率。这一发现表明，所观察到的效应不能完全归因于CA-EO或LP-EO，并提示载体基质或其组分产生的理化条件对细菌生长抑制有所贡献。因此，对MIC和CFU结果的解释应考虑完整制剂，而非仅考虑EO浓度。综上所述，琼脂扩散与系列稀释结果之间的差异强调了实验系统对复杂外用制剂表观抗菌性能的影响。先前研究已证明不同EO对葡萄球菌具有抗菌活性，包括对甲氧西林敏感和耐甲氧西林的假中间葡萄球菌分离株(46, 47)，但本研究结果提示，掺入干粉基质可能改变对分离油所观察到的抗菌行为。因此，当前发现提供了在特定体外条件下抗菌活性的初步证据，而非对成品香波抗菌功效的确定性确认。进一步研究应评价更大的临床分离株集合，并纳入补充方法，如制剂释放研究、时间-杀菌试验和抗生物膜模型，以更清晰地区分EO和载体基质对所观察抗菌反应的贡献。4.4 小鼠模型中的安全性试验在宏观理化表征之后，对三种最终制剂(SLP、SCA和SVE)进行了体内测试，以评估其初步皮肤安全性。为此使用了小鼠模型。4.4.1 临床前安全性评估在五天实验期内对代表性动物进行摄影监测(图4)，可评价应用部位的皮肤反应。在整个研究期间，皮肤保持完整，无可见刺激迹象，包括红斑、水肿、溃疡、渗出物、严重脱屑、开放性病变、结痂、出血或坏死。个体间观察到的轻微皮肤颜色变化不一致或非进行性，因此归因于正常生理差异或实验操作过程中的处理(18, 20)。一般福利参数——包括外观、食物和水摄入、体温、呼吸和心率以及自发行为——在整个观察期内保持在正常范围内。体重波动极小，通常低于5%，且在动物或治疗组之间未显示一致趋势，这符合幼年动物适应实验条件时的正常生理变异(17, 19)。4.4.2 组织学分析试验结束时采集的皮肤样本的组织学检查显示，SLP、SCA和SVE组的组织结构保持完好(图5)，表皮连续，真皮层界限清晰。无溃疡、糜烂、
Varas等，10.3389/fvets.2026.1918882，《前沿兽医科学》，18，frontiersin.org。未观察到坏死或增生。真皮显示正常的胶原纤维组织结构，无炎症浸润或水肿的证据，在所评估的条件下无治疗相关细胞损伤或炎症反应的组织学证据。在所有组中均观察到可比的组织学特征，包括赋形剂制剂组，提示洗发水基质本身在所评估条件下未产生可检测的组织改变。特别是，含精油（EO）的制剂（SLP和SCA）表现出与赋形剂相似的组织反应，提示在测试条件下加入EO未引入组织学改变。结合临床观察，这些发现提供了初步证据，表明所开发的 prototypes 在所评估的局部暴露条件下耐受良好，并支持针对潜在兽医应用进一步开展皮肤安全性评价（18, 20, 48）。在BALB/c小鼠中进行的临床前皮肤安全性评价提供了额外证据，支持制剂的耐受性。重复局部给药在所评估条件下未产生明显的临床不良体征、行为改变、红斑、水肿或炎症或组织损伤的组织学证据。这一发现具有相关性，因为EOs可能根据其化学组成和浓度对哺乳动物细胞发挥生物学效应；因此，安全性评估是开发基于EO的局部产品的重要步骤（49）。然而，在小鼠中获得的结果应视为初步临床前证据，不能替代犬患者在临床条件下的安全性评价。本研究的另一个局限性是未直接评估真皮渗透和EO成分的全身暴露；因此，当前发现无法确定局部给药后的经皮吸收程度。尽管监测了局部皮肤耐受性和一般安全性参数，但这些评估不能替代专门的渗透或药代动力学研究。因此，未来研究应包括使用切除犬皮肤和扩散池系统的体外/离体渗透实验，遵循OECD测试指南428的原则，从而定量测定保留在皮肤内和到达受体室的代表性EO成分，进而提供关于真皮滞留和经皮吸收的信息。还应纳入暴露前后皮肤屏障完整性的评估，以确定重复给药是否影响皮肤屏障功能，同时对重复局部给药后血浆中代表性EO成分进行补充定量将有助于表征全身暴露和生物利用度。总之，这些方法对于确定暴露是否主要局限于局部以及更全面地表征制剂在重复局部使用下的安全性特征将很重要。4.5 犬的微生物学评价。在犬中进行的微生物学评估为干洗香波制剂在临床条件下的抗菌行为提供了初步的体内近似评价。为更全面地表征反应，考虑了三个结局：细菌存在或不存在、细菌载量类别以及生长被分类为显著或不显著。治疗前，几乎所有犬均培养出葡萄球菌属阳性，支持病变的感染性质，并与公认的伪中间葡萄球菌在犬浅表脓皮病中的优势地位一致（2, 50）。至第21天，SCA和Inveclor ®组记录到培养中无可检测球菌，而SLP和SVE组中所有可评估犬仍为培养阳性。最高清除频率见于Inveclor ®组，考虑到基于氯己定的局部产品在犬浅表脓皮病治疗中的既定作用，这是预期之中的（2, 51）。在载有精油的干洗香波中，SCA是唯一观察到培养阴性的含EO制剂，提示C. alba EO制剂具有更有利的基于培养的反应。细菌载量分类使反应能够以比单纯培养阳性更高的分辨率进行解读。在第21天，SCA组中归入非显著细菌载量类别的犬比例最大，其次为Inveclor ®、SVE和SLP。就SLP组而言，一只犬在第21天显示非显著细菌生长，而其余病例观察到显著细菌生长。此外，SLP组中不可计数类别的犬比例最高，这提示先前在体外观察到的L. philippiana EO抗菌活性未能在基于培养的体内评价中明确重现。将细菌生长分类为显著或不显著支持了这一趋势，SCA在第21天显示非显著细菌生长的犬比例最高。然而，治疗组之间的比较未揭示统计学显著差异。因此，这些结果应解释为探索性趋势，而非治疗疗效的确定性证据。尽管如此，细菌载量和生长分类可能代表未来研究的有用终点，因为它们允许在二分类存在/不存在结局之外评价微生物学反应。这种区分具有临床相关性，因为犬浅表脓皮病的治疗并不完全依赖于完全根除细菌。减少细菌负荷、限制病变进展、改善皮肤微环境和支持屏障恢复也是重要的治疗目标。当前建议强调以细胞学支持的诊断、必要时进行细菌培养、针对表面和浅表感染的局部治疗以及负责任的抗菌药物使用，以减少不必要的全身抗生素暴露（2, 6）。在此框架内，有助于减少细菌负荷的局部产品即使在并非每位患者均实现完全微生物清除的情况下也可能有价值。SCA观察到的相对更好的微生物学特征可能与C. alba EO的化学组成有关。该精油含有单萜类如桧烯、桉叶油醇、松油烯-4-醇、α-蒎烯和β-蒎烯，这些成分与涉及膜破坏、细胞通透性增加、细胞内成分泄漏以及干扰细菌黏附或持续存在的抗菌效应有关（10, 14, 52）。针对葡萄球菌测试的其他精油也报道了类似机制，包括与犬脓皮病相关的伪中间葡萄球菌分离株（46, 47）。
Varas等，10.3389/fvets.2026.1918882，《前沿兽医科学》，19，frontiersin.org。成品制剂体外性能的测定方法依赖性差异与体内获得的微生物学应答之间的差异，进一步说明将实验室抗菌结果外推至临床环境存在困难。在犬中，细菌动态受病变特征、皮肤屏障状况、局部炎症、宿主免疫、梳理行为、环境暴露、主人依从性以及制剂在皮肤上的滞留情况影响。这些变量在浅表性脓皮病中尤为重要，该病通常继发于屏障功能障碍、过敏性疾病或复发性皮肤病（50, 53）。因此，本犬试验应被视为探索性和假设生成性研究，而非确定性研究。这些结果还应在抗菌药物耐药性的更广泛背景下解读。假中间葡萄球菌是犬的关键病原体，耐甲氧西林或多重耐药菌株仍是兽医学中的主要关注问题（1, 54, 55）。在本研究中，所有回收的假中间葡萄球菌分离株均未显示对苯唑西林的表型耐药。然而，该发现应谨慎解读，因为甲氧西林耐药性是通过表型评估的，并未通过mecA等耐药决定因素的分子检测加以确认。因此，结果表明未检出表型MRSP，而非确定排除甲氧西林耐药性。因此，开发耐受性良好的局部替代制剂可能有助于以“同一健康”为导向的策略，减少对全身抗菌药物的依赖，尤其考虑到假中间葡萄球菌潜在的人畜共患传播（3, 56）。应承认若干局限性。尽管最终干香波制剂的抗菌活性通过琼脂扩散、倍比稀释、MIC评估和定量活菌计数直接评价，但这些分析为初步性，仅包括假中间葡萄球菌ATCC 49444和三株临床分离株。因此，观察到的抗菌应答尚不能被视为代表与犬浅表性脓皮病相关的更广泛细菌群体（46, 47）。此外，由于SVE在若干相应制剂稀释水平下也减少细菌回收，不能排除载体基质的贡献，观察到的体外活性不能完全归因于所加入的EOs。该临床研究旨在检测较大的微生物学效应，但检验效能不足以检测小或中等治疗效应，或确立明确的临床疗效、等效性或非劣效性。此外，微生物学结局未与标准化定量病变严重程度评分整合，EO成分的个体贡献或潜在协同相互作用未通过实验确定。其他局限性涉及入组犬群体的临床异质性。犬在真实世界条件下从多个兽医中心招募，相当比例为混种动物。尽管入选标准要求全身或局部抗生素、皮质类固醇以及具有潜在抗菌或抗炎活性的天然产品至少15天无治疗期，但并非所有动物均可获得关于品种或品种类型以及该排除期之前接受的皮肤病治疗的完整标准化信息。因此，这些变量无法纳入治疗组分层或统计调整，可能促成微生物学应答的变异性。此外，临床随访仅限于21天治疗期；因此，无法确定微生物学应答的持续性、浅表性脓皮病的复发以及治疗停止后的长期耐受性。因此，未来研究应包括更大且更多样化的临床分离株收集、补充性的时间-杀菌、抗生物膜、制剂释放和皮肤滞留研究，以及使用标准化临床结局指标的更大规模随机对照临床试验（2, 44, 50）。总体而言，尽管成品制剂的抗菌性能具有测定方法依赖性，且体内观察到的组间微生物学差异无统计学显著性，但将天然植物EOs纳入具有初步物理稳定性和良好临床前耐受性的干香波平台，代表了制剂开发的相关步骤。在含EO制剂中，SCA显示出最有利的探索性微生物学特征，达到无显著细菌负荷的犬比例最高，并且是载EO香波组中唯一观察到培养阴性病例的组。这些发现支持进一步研究含C. alba EO的干香波作为犬浅表性脓皮病的潜在局部候选制剂，同时强调在就临床疗效得出结论之前需要更大规模的对照研究（2, 23）。5 结论。本研究报道了掺入L. philippiana和C. alba EOs的淀粉基干香波原型的开发与表征，并评估了其体外抗菌性能、临床前皮肤耐受性，以及在自然发生假中间葡萄球菌相关浅表性脓皮病犬中的探索性微生物学应答。总体而言，不同实验阶段针对的核心假设是：这些EOs可被纳入干香波制剂，具有初步物理稳定性、在所评价条件下可接受的局部皮肤耐受性以及可测量的微生物学应答。使用智利本土植物作为生物活性化合物来源，代表了开发兽医皮肤病学补充性局部方法的一种潜在策略。从制剂角度看，加入硬脂酸镁并选择适当的混合方案改善了粉末均匀性，减少了团聚，并增强了处理和流动相关特性。补充的理化表征进一步表明，所选原型仍为黏聚性粉末系统，并在所评价条件下表现出初步物理稳定性。然而，碱性pH、缺乏批间重现性评估以及缺乏纵向化学谱分析仍是相关的制剂局限性。对成品制剂的直接体外评价揭示了测定方法依赖性的抗菌行为。无可测量的抑制作用
Varas等，10.3389/fvets.2026.1918882，Frontiers in Veterinary Science，20frontiersin.org。通过琼脂扩散法观察到抑菌圈，而连续稀释和活菌计数实验显示细菌回收率呈浓度依赖性降低。由于赋形剂配方也观察到抑制作用，因此抗菌反应不能完全归因于所加入的精油。这些发现强调了评估完整配方而非仅从分离的精油外推抗菌活性的重要性。临床前安全性评估显示，在BALB/c小鼠中重复局部施用含精油配方和赋形剂，在所评估条件下未产生可见红斑、水肿、相关行为或体重改变，也无治疗相关炎症或组织损伤的组织学证据。这些发现提供了局部皮肤耐受性的初步证据，但不能替代目标物种的安全性评估或专门的皮肤渗透和全身暴露研究。在浅表性脓皮病犬中，治疗组间在细菌检出、细菌载量类别以及显著与非显著生长方面观察到数值差异。这些差异无统计学显著性，不应解释为临床疗效的证据。尽管如此，在含精油配方中，SCA显示出最有利的探索性微生物学特征，包括达到非显著细菌载量的犬比例最高，以及含精油香波组中唯一观察到的培养阴性病例。免洗干粉剂型可能对局限性病变、间擦部位或难以或不适合反复洗澡的犬特别有利，可能简化反复局部给药并减少操作要求。然而，这些实际优势在本研究中未经过正式评估，应在未来的可用性和主人依从性研究中予以确认。综上所述，这些发现支持将L. philippiana和C. alba精油纳入淀粉基干香波原型的可行性，并提供了物理稳定性、临床前皮肤耐受性以及检测方法依赖性抗菌和探索性微生物学性能的初步证据。免洗粉剂型可能提供实际优势，包括局部施用和无需冲洗给药，尽管这些潜在益处未经正式评估。进一步工作应包括pH优化、独立制备的配方批次、定量纵向化学稳定性分析、配方释放、时间杀菌和抗生物膜研究、皮肤渗透和皮肤滞留评估、纳入经验证临床评分系统的更大规模随机对照临床试验，以及在得出临床疗效或长期兽医使用结论之前进行扩展安全性评估。6 专利。抗菌干香波的国家注册申请已提交至智利INAPI，目前正在审查受理中。数据可用性声明。本研究呈现的原始贡献包含在文章/补充材料中。进一步咨询可联系通讯作者。伦理声明。动物研究经智利大学牙学院伦理委员会批准（方案24814-ODO-UCH-e3；2025年6月4日）和Mayor大学伦理委员会批准（方案ID14/2024；2024年6月28日）。研究按照当地法规和机构要求进行。已获得动物主人书面知情同意，同意其动物参与本研究。作者贡献。MV：数据管理、形式分析、调查、方法学、可视化、撰写—原稿、撰写—审阅与编辑。MJ：数据管理、形式分析、调查、方法学、可视化、撰写—原稿、撰写—审阅与编辑。NR：数据管理、形式分析、调查、方法学、撰写—原稿、撰写—审阅与编辑。PR：调查、方法学、项目管理、资源、撰写—原稿。FB：概念化、方法学、撰写—原稿。GV-B：概念化、验证、可视化、撰写—审阅与编辑。OC：概念化、验证、撰写—原稿。AD-P：调查、方法学、撰写—原稿。MA：形式分析、撰写—审阅与编辑。GM：数据管理、形式分析、可视化、撰写—原稿、撰写—审阅与编辑。IM：可视化、撰写—审阅与编辑。FA：调查、方法学、验证、撰写—原稿。BR：调查、方法学、验证、撰写—原稿。MB：调查、方法学、验证、撰写—原稿。DS：概念化、项目管理、验证、可视化、撰写—审阅与编辑。JB：概念化、形式分析、资金获取、调查、项目管理、资源、监督、验证、可视化、撰写—原稿、撰写—审阅与编辑。资金。作者声明本研究工作和/或其发表获得了财政支持。本工作由IDEA ID24I10250；INGE 230004；CIA250002–CEDENNA资助。致谢。作者感谢Oftaderm兽医诊所、Mayor大学兽医教学医院以及La Granja市立兽医诊所的兽医团队。
Varas等人（10.3389/fvets.2026.1918882，《兽医科学前沿》21 frontiersin.org）以及Conchalí（智利圣地亚哥）在纳入初步微生物学评估的犬类患者的招募和临床管理方面给予了宝贵合作。他们的支持与投入对本研究临床部分的开展至关重要。利益冲突：FA和BR受雇于Oftaderm兽医诊所。其余作者声明，本研究是在不存在任何可被解释为潜在利益冲突的商业或财务关系的情况下进行的。生成式人工智能声明：作者声明在本稿件的创作中使用了生成式人工智能。作者承认在稿件准备期间使用了由OpenAI开发的基于GPT-5.5 Thinking的ChatGPT，用于语言编辑、语法修订和格式支持。所有人工智能辅助的输出均由作者审阅和核实。作者对最终稿件的内容、准确性、原创性和完整性承担全部责任。本文图表旁提供的任何替代文本（alt text）均由Frontiers在人工智能的支持下生成，并已尽合理努力确保准确性，包括在可能的情况下由作者进行审阅。如果您发现任何问题，请联系我们。出版商声明：本文中表达的所有主张仅代表作者本人，不一定代表其所属组织，也不一定代表出版商、编辑和审稿人的观点。本文中可能评估的任何产品或其制造商可能提出的任何主张，均不受出版商的保证或认可。补充材料：本文的补充材料可在线获取，网址为：https://www.frontiersin.org/articles/10.3389/fvets.2026.1918882/full#supplementary-material 参考文献 1. Pires dos Santos T, Damborg P, Moodley A, Guardabassi L. 耐甲氧西林假中间葡萄球菌全球流行病学系统综述：基于多位点序列分型数据的种群结构推断。Front Microbiol. (2016) 7:1599. doi: 10.3389/fmicb.2016.01599 2. Loeffler A, Cain CL, Ferrer L, Nishifuji K, Varjonen K, Papich MG, 等. 国际伴侣动物传染病学会（ISCAID）犬脓皮病抗菌药物使用指南。Vet Dermatol. (2025) 36:234–82. doi: 10.1111/vde.13342 3. Moses IB, Santos FF, Gales AC. 假中间葡萄球菌的人类定植与感染：一种新发且被低估的人畜共患病原体。Microorganisms. (2023) 11:581. doi: 10.3390/microorganisms11030581 4. Viktorová J, Kumar R, Řehořová K, Hoang L, Ruml T, Figueroa CR, 等. 两种智利本土水果提取物的抗菌活性：Arrayan（Luma apiculata）和Peumo（Cryptocarya alba）。Antibiotics. (2020) 9:444. doi: 10.3390/antibiotics9080444 5. Zoccolotti J d O, Cavalheiro AJ, Tasso CO, Ribas BR, Ferrisse TM, Jorge JH. Cryptocarya物种提取物的抗菌功效与生物相容性。PLoS One. (2021) 16:e0261884. doi: 10.1371/journal.pone.0261884 6. Hillier A, Lloyd DH, Weese JS, Blondeau JM, Boothe D, Breitschwerdt E, 等. 犬浅表细菌性毛囊炎诊断与抗菌治疗指南（国际伴侣动物传染病学会抗菌指南工作组）。Vet Dermatol. (2014) 25:163–e43. doi: 10.1111/vde.12118 7. Cheng F, Ma X, Lu X, Zhu Y, Abula R, Wu T, 等. 从Schizonepeta annua提取的精油通过膜破坏对耐甲氧西林金黄色葡萄球菌的抗菌特性。Microb Pathog. (2024) 196:106975. doi: 10.1016/j.micpath.2024.106975 8. Meenu M, Padhan B, Patel M, Patel R, Xu B. 植物不同部位精油对沙门菌和李斯特菌的抗菌活性。Food Chem. (2023) 404:134723. doi: 10.1016/j.foodchem.2022.134723 9. Yadav A, Paul KK, Soni M, Dubey NK, Prasad R, Tilak R, 等. 皮肤癣菌的抗真菌耐药性：基于精油的纳米制剂作为抗皮肤癣菌的新一代疗法。Microb Pathog. (2025) 205:107622. doi: 10.1016/j.micpath.2025.107622 10. de Sousa DP, Damasceno ROS, Amorati R, Elshabrawy HA, de Castro RD, Bezerra DP, 等. 精油：化学与药理活性。Biomolecules. (2023) 13:1144. doi: 10.3390/biom13071144 11. Bolgen UMG, Demirci Kayiran S, Ozogul Y, Ozogul F. 基于精油的纳米乳研究现状：配方、表征及在食品和药物中的应用。Ind Crop Prod. (2025) 233:121411. doi: 10.1016/j.indcrop.2025.121411 12. Castro-Saavedra S, Fuentes-Barros G, Tirapegui C, Acevedo-Fuentes W, Cassels BK, Barriga A, 等. 智利特有树种Cryptocarya alba生物碱的植物化学分析。J Chil Chem Soc. (2016) 61:3076–80. doi: 10.4067/S0717-97072016000300014 13. Bruna F, Fernández K, Urrejola F, Touma J, Navarro M, Sepúlveda B, 等. 智利Laureliopsis philippiana精油的化学成分、抗氧化、抗菌及抗增殖活性：体外与计算机模拟研究。Arab J Chem. (2022) 15:104271. doi: 10.1016/j.arabjc.2022.104271 14. Touma J, Navarro M, Sepúlveda B, Pavon A, Corsini G, Fernández K, 等. 源自Cryptocarya alba和Laurelia sempervirens的精油化学成分具有抗氧化、抗菌和抗肿瘤活性潜力。Molecules. (2020) 25:5600. doi: 10.3390/molecules25235600 15. Aiemsaard J, Kamollerd C, Seubsasana S, Thongkham E, Vonghataipaisarn P. 柠檬草精油增强头孢氨苄对分离自犬浅表脓皮病的假中间葡萄球菌的抗菌活性。ScienceAsia. (2021) 47:690. doi: 10.2306/scienceasia1513-1874.2021.093 16. 欧洲议会. 欧洲议会和理事会2018年12月11日关于兽药产品并废止第2001/82/CE号指令的条例（欧盟）2019/6（与欧洲经济区相关的文本）。可在线获取：http://data.europa.eu/eli/reg/2019/6/oj（2026年6月13日访问）。17. 农业与畜牧业服务局. 指南：申请人提交药物产品注册档案所执行的活动。(2022)。可在线获取：https://www.sag.gob.cl/sites/default/files/Gu%C3%ADa%20Registro%20Productos%20Farmacol%C3%B3gicos_v10.pdf（2025年12月26日访问）。18. OECD. 化学品测试指南。急性皮肤刺激/腐蚀。(2015)。可在线获取：https://www.oecd.org/content/dam/oecd/en/publications/reports/2015/07/test-no-404-acute-dermal-irritation-corrosion_g1g59b23/9789264242678-en.pdf 19. Nelson M, Barnes KB, Davies CH, Cote CK, Meinig JM, Biryukov SS, 等. 用于评估气溶胶化 Burkholderia pseudomallei 感染治疗方法的BALB/c小鼠模型。Antibiotics. (2023) 12:506. doi: 10.3390/antibiotics12030506 20. Morton DB, Griffiths PH. 实验动物疼痛、痛苦和不适识别指南及评估假说。Vet Rec. (1985) 116:431–6. doi: 10.1136/vr.116.16.431 21. Prescott D, Stewart A, Schoep A, Herndon A. 含精油、锌和水杨酸的局部乳膏可减轻瘙痒犬的瘙痒和皮肤病变评分。Aust Vet J. (2026) 104:196–205. doi: 10.1111/avj.70048
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Frontiers in Veterinary Science01frontiersin.org TYPE Original Research PUBLISHED 05 October 2026 DOI 10.3389/fvets.2026.1918882 OPEN ACCESS EDITED BY Benito Soto-Blanco, Federal University of Minas Gerais, Brazil REVIEWED BY Hilke Oltmanns, University of Veterinary Medicine Hannover, Germany Ujban Hussain, Rashtrasant Tukadoji Maharaj Nagpur University, India *CORRESPONDENCE Daniela Siel daniela.siel@umayor.cl Jessica Bravo jessica.bravo@mail.udp.cl RECEIVED 25 June 2026 REVISED 10 August 2026 ACCEPTED 24 August 2026 PUBLISHED 05 October 2026 CITATION Varas M, Jacobs M, Rojas N, Ramos P, Bruna F, Valenzuela-Barra G, Correa O, Díaz-Parra A, Ahumada M, Maturana G, Martínez I, Abusleme F, Rivera B, Bargsted MO, Siel D and Bravo J (2026) Stability and dermatological safety assessment of Cryptocarya alba (Peumo) and Laureliopsis philippiana (Tepa) essential oil-carrying dry shampoo formulations for dermal use in dogs. Front. Vet. Sci. 13:1918882. doi: 10.3389/fvets.2026.1918882 COPYRIGHT © 2026 Varas, Jacobs, Rojas, Ramos, Bruna, Valenzuela-Barra, Correa, Díaz-Parra, Ahumada, Maturana, Martínez, Abusleme, Rivera, Bargsted, Siel and Bravo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Stability and dermatological safety assessment of Cryptocarya alba (Peumo) and Laureliopsis philippiana (Tepa) essential oil-carrying dry shampoo formulations for dermal use in dogs Martín Varas 1,2 , Martina Jacobs 1,2 , Nicole Rojas 1 , Paola Ramos 1 , Flavia Bruna 1,3,4 , Gabriela Valenzuela-Barra 1,5 , Olosmira Correa 1,5 , Antonia Díaz-Parra 1,6 , Manuel Ahumada 6 , Gabriela Maturana 1 , Irene Martínez 2 , Francisco Abusleme 7,8 , Belén Rivera 8 , María Olga Bargsted 7 , Daniela Siel 7,9 * and Jessica Bravo 1 * 1 Laboratory of Bioactive Natural Products, School of Medicine, Center for Biomedical Research, Diego Portales University, Santiago, Chile, 2 Department of Chemical Engineering, Biotechnology and Materials, Faculty of Physical and Mathematical Sciences, University of Chile, Santiago, Chile, 3 Hormone and Cancer Biology Laboratory, Institute of Experimental Medicine and Biology of Cuyo (IMBECU), CONICET CCT Mendoza—National University of Cuyo (UNCuyo), Mendoza, Argentina, 4 Center of Odontological Research (CIO) From the Faculty of Odontology, National University of Cuyo (UNCuyo), Mendoza, Argentina, 5 Department of Pharmaceutical Sciences and Technology, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile, 6 School of Biotechnology, Faculty of Sciences, Engineering and Technology, Mayor University, Santiago, Chile, 7 School of Veterinary Medicine, Faculty of Medicine and Health Sciences, Mayor University, Santiago, Chile, 8 Oftaderm Veterinary Clinic, Santiago, Chile, 9 Center for Biomedicine, Mayor University, Santiago, Chile Introduction: Antibiotic resistance is a major concern in human and veterinary medicine, supporting the development of alternative topical products for companion animals. This study aimed to develop and evaluate essential oil (EO)- based dry shampoo formulations incorporating EOs from Cryptocarya alba and Laureliopsis philippiana as potential veterinary dermocosmetic platforms for canine skin care and complementary management of superficial pyoderma. Methods: Starch-based dry shampoo prototypes containing C. alba or L. philippiana EOs were developed using corn starch, tapioca starch, or their mixtures. Magnesium stearate (MgS) was evaluated as a functional excipient, and two mixing protocols were compared. Optimized formulations were subjected to physicochemical characterization, thermal, mechanical, and 9-month stability assessments, and in vitro antibacterial testing against Staphylococcus pseudintermedius. Preclinical dermal safety was evaluated in BALB/c mice through macroscopic irritation scoring, body weight monitoring, and histological analysis. Subsequently, an exploratory, randomized, controlled, blinded, four-arm parallelgroup study was conducted in 32 client-owned dogs with superficial pyoderma allocated to receive SLP, SCA, vehicle shampoo, or a chlorhexidine-based reference treatment for 21 days. Results: MgS improved formulation homogeneity, fluidity, and reduced agglomeration, while the selected mixing protocol enhanced overall powder performance. The
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science02frontiersin.org optimized formulations showed preliminary physical stability, although progressive changes in texture, fluidity, agglomeration tendency, and aroma were observed during storage. In vitro antibacterial activity was assay-dependent: no measurable inhibition zones were observed by agar diffusion, whereas serial dilution and viablecount assays showed concentration-dependent reductions in bacterial recovery, including effects associated with the vehicle formulation. No macroscopic or histological signs of dermal irritation were observed in mice. In dogs, the formulations were well tolerated under the evaluated conditions. Microbiological outcomes differed numerically among groups but not significantly; among the EO-containing formulations, SCA showed the most favorable exploratory profile, including culture negativity in 3/8 dogs and the highest proportion of dogs reaching a non-significant bacterial burden. Discussion: These findings support the feasibility of incorporating C. alba and L. philippiana EOs into starch-based dry shampoo prototypes and provide preliminary evidence of physical stability and dermal tolerability. However, the assay-dependent antibacterial response, alkaline formulation pH, limited batch replication, and exploratory clinical design preclude conclusions regarding clinical efficacy. Further studies should include formulation optimization, quantitative chemical stability assessment, dermal permeation studies, and larger randomized controlled clinical trials. KEYWORDS essential oils, Cryptocarya alba, Laureliopsis philippiana, dry shampoo, canine superficial pyoderma, Staphylococcus pseudintermedius 1 Introduction Pyoderma is among the most common health problems in dogs and is often caused by bacteria, with Staphylococcus pseudintermedius being the most frequent agent (1). The traditional treatment for pyoderma includes antibiotics (2), but antibiotic misuse is now one of the main causes of bacterial resistance, a major concern for human and animal health. New antibiotic agents are urgently needed to treat multidrug-resistant bacteria (3). Topical pharmaceutical formulations are recommended for treatment of superficial pyoderma even in the presence of methicillin-resistant strains (2). Previous studies have explored alternative topical strategies for canine superficial pyoderma. For example, cyclodextrin-based protocols have shown efficacy comparable to systemic antibiotic therapy in selected cases, highlighting the potential of locally applied formulations to reduce reliance on systemic antimicrobials (4). However, the performance of topical therapies depends on multiple formulation-related factors, including the concentration of the active compound, surfactant use, pH, application frequency, and contact time (5, 6). Two to three applications per week with contact times between 5 and 10 min are recommended, but studies have shown that long-term treatments may disrupt the normal protective function of the dermis, which is especially relevant in patients with previously inflamed skin (6). Plant EOs are complex mixtures containing terpenes, terpenoids, and phenyl terpenes, among other bioactive compounds (7, 8). Plants synthesize these secondary metabolites as a defense mechanism against pathogens, and due to their antioxidant, antiinflammatory, antibacterial, and antifungal properties (7–10), EOs have been studied for their application in the pharmaceutical industry (11). Cryptocarya alba (Peumo) and Laureliopsis philippiana (Tepa) are two tree species native to Chile that the Mapuche people have used to treat liver diseases, rheumatism, and wounds (12, 13). The EOs obtained from C. alba and L. philippiana are rich in 1,8-cineole, also known as eucalyptol, linalool, and safrole, among other compounds (13, 14), and their antibacterial activity has been reported against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, among others (13, 14). For veterinary topical formulations intended for repeated application, in vivo assessment is essential to establish both safety and biological performance before clinical use. Murine models are widely used to evaluate local dermal tolerance, including erythema, edema, histological alterations, and potential systemic effects, whereas studies in dogs, as the target species, allow preliminary evaluation of clinical and microbiological responses under real dermatological conditions (15–22). This study hypothesized that essential oils from C. alba and L. philippiana could be incorporated into a starch-based dry shampoo with acceptable physical stability and local dermal tolerability, and that the resulting formulations could produce measurable microbiological responses in dogs with superficial pyoderma. Therefore, the main objective was to develop and characterize EO-loaded dry shampoo Abbreviations: CA, Cryptocarya alba; CA-EO, Cryptocarya alba essential oil; EO, essential oil; LP, Laureliopsis philippiana; LP-EO, Laureliopsis philippiana essential oil; MgS, magnesium stearate; SCA, shampoo formulation carrying CA-EO; SLES, sodium lauryl ether sulfate; SLP, shampoo formulation carrying LP-EO; SLP1, formulation with corn starch and LP-EO; SLP1-MgS, formulation with corn starch, MgS, and LP-EO; SLP2, formulation with tapioca and LP-EO; SLP2-MgS, formulation with tapioca, MgS, and LP-EO; SLP3, formulation with mixture of starch sources and LP-EO; SLP3-MgS, formulation with mixture of starch sources, MgS, and LP-EO; SVE, shampoo vehicle.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science03frontiersin.org prototypes, assess their preclinical dermal safety, and evaluate their microbiological performance in client-owned dogs with superficial pyoderma. 2 Materials and methods The experimental workflow of the study is summarized in Figure 1. Briefly, the methodology comprised five sequential stages: plant sampling and essential oil extraction, dry shampoo formulation development, physicochemical characterization, preclinical safety testing in mice, and clinical evaluation in dogs. Each stage was designed to progressively assess formulation feasibility, stability, safety, and microbiological performance. 2.1 Sampling and extraction Fresh leaves of C. alba (CA) were collected during spring (November 2024) at the end of the flowering period and before fruit formation, in Chicauma, Lampa, Metropolitan Region, Chile (33°12′3.8808′′S, 70°58′8.1156′′W). Similarly, fresh leaves of L. philippiana (LP) were collected in October 2024 during the anthesis period in Los Pellines, Valdivia, Los Ríos Region, Chile (39°44′42.0′′S, 73°21′25.2′′O). Voucher specimens were prepared for both species. CA (SQF 22901) was deposited in the Herbarium of the Faculty of Chemistry and Pharmacy, University of Chile, while LP (VALD 3560) was deposited in the Herbarium of the Universidad Austral de Chile. Plant material was processed for extraction on the day of collection or within 24 h thereafter. EO extraction from both species was carried out immediately after collecting using a 10-L Clevenger-type apparatus via steam distillation. The process was conducted in batches until the entire plant material was distilled (1 h per batch in both cases). The resulting heterogeneous mixture was separated using a separatory funnel to recover the EO. The hydrosol was discarded. Thus, two EO were obtained: CA essential oil (CA-EO) and LP essential oil (LP-EO). The chemical profile of these essential oils has been previously characterized by gas chromatography–mass spectrometry (GC–MS) by Jacobs et al. (23). In that analysis, a total of 62 volatile compounds were detected across both oils. CA-EO showed a more complex profile, with 43 identified compounds, among which sabinene and eucalyptol were the major constituents, representing 13.95 and 12.05% of the total chromatographic area, respectively. In contrast, LP-EO presented 20 identified compounds, with linalool, eucalyptol, and safrole as the predominant constituents, accounting for 37.91, 24.54, and 11.42% of the total chromatographic area, respectively. These findings confirm that CA-EO and LP-EO exhibit clearly distinct phytochemical profiles, which may contribute to differences in their biological and antibacterial properties. The in vitro antibacterial activity of CA-EO and LP-EO against Staphylococcus pseudintermedius has also been previously reported by Jacobs et al. (23). In that study, both essential oils were evaluated against 12 S. pseudintermedius isolates from dogs with pyoderma, together with the reference strain S. pseudintermedius ATCC 49444, using agar diffusion and minimum inhibitory concentration (MIC) assays. Both oils showed moderate antibacterial activity, with inhibition zones ranging from 4.92 to 10.80 mm. CA-EO showed a higher mean inhibition diameter than LP-EO, with average values of 9.09 mm and 6.76 mm, respectively, corresponding to 37 and 28% of the activity observed for the reference antiseptic chlorhexidine. The most susceptible strains were SP9 for CA-EO and SP13 for LP-EO, with inhibition zones of 10.38 and 10.80 mm, respectively. MIC values were generally ≥4.096 mg mL −1 for both oils, except for LP-EO against strain SP12, which showed an MIC of 2.048 mg mL −1 . These results support the antibacterial potential of both essential oils FIGURE 1 Experimental workflow of the study. The methodological sequence included plant sampling and essential oil (EO) extraction, formulation development, physicochemical characterization, preclinical safety testing in BALB/c mice, and clinical evaluation in dogs. CA-EO: Cryptocarya alba essential oil; LP- EO: Laureliopsis philippiana essential oil; MgS: magnesium stearate; SCA: dry shampoo containing C. alba essential oil; SLP: dry shampoo containing L. philippiana essential oil; SVE: vehicle formulation; FTIR: Fourier-transform infrared spectroscopy; DSC: differential scanning calorimetry; SEM: scanning electron microscopy; CFU: colony-forming units.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science04frontiersin.org against S. pseudintermedius, although with lower activity than chlorhexidine. 2.2 Formulation and homogeneity test To evaluate homogeneity, six shampoo formulations carrying LP-EO (SLP) were prepared in 10 g samples designed considering commercial dry shampoo formulation examples from AGRANA STARCH 1 and AkzoNobel Surface Chemistry. 2 To evaluate homogeneity, six shampoo formulations carrying LP-EO (SLP) were prepared in 10 g samples based on commercial products (AGRANA – MAISITA 9040 and Zephyrous Dry Shampoo 2,906–100) and taking as reference previous work on dry herbal powder shampoo formulations (24). Three starches were tested: cornstarch, tapioca, and a mixture of both. Magnesium stearate was selected because of its established use in solid pharmaceutical and cosmetic formulations and its reported influence on powder flow, particle cohesion, and agglomeration. These properties were considered particularly relevant for improving the incorporation and distribution of the lipophilic EO within the starch-based matrix. To evaluate its functional contribution, otherwise comparable formulations were prepared with and without MgS (25–27). Formulations were grouped according to the starch used, and each of these groups was subdivided into two, depending on the addition of MgS, resulting in the 6 formulations: formulation based on cornstarch without MgS (SLP1) and with MgS (SLP1-MgS), formulation based on tapioca without MgS (SLP2), and with MgS (SLP2-MgS), formulation based on a mixture of cornstarch and tapioca without MgS (SLP3), and with MgS (SLP3-MgS). For visual assessment of EO dispersion, LP-EO was previously mixed with Sudan Red B (Merck, Chile), an oil-soluble dye. The starch was combined with MgS when applicable, followed by the addition of Sudan Red B–colored LP-EO. The mixture was agitated in an Incu-Shaker™ Mini at 200 rpm for 18 h, ground with a mortar and pestle to break agglomerates. Sodium lauryl ether sulfate (SLES) 1 AGRANA STARCH. Non-aerosol Dry Shampoo with MAISITA 9040 and TAPIOCA NATURAL. Available at: https://www.agrana.com/downloadcenter?tx_ thagranadownloadcenter_downloadcenter%5Baction%5D=download&tx_thagranadownloadcenter_downloadcenter%5Bcontroller%5D=Downloadcente r&tx_thagranadownloadcenter_downloadcenter%5Bdownload%5D=4856&t ype=97687851&cHash=6f68fc458981b7bde90b514c1f5f7bcf 2 AkzoNobel Surface Chemistry. Zephyrous Dry Shampoo. Available at: https://www.essentialingredients.com/pdf/AkzoNobel%20Zephyrous%20 Dry%20Shampoo%20%282906-100%29.pdf was subsequently incorporated as an anionic surfactant to improve the wetting and dispersion of the hydrophobic EO within the powder matrix and to provide a cleansing function consistent with the intended use of the formulation as a topical dry shampoo (28). The mixture was then agitated for an additional 2 h (Figure 2). This mixing method will be referred to as Method A. The resulting powders were evaluated using a qualitative scale based on seven organoleptic and physical parameters (Table 1), including homogeneity, texture, color, fluidity, and odor, scored on a 1 to 5 qualitative scale designed according to general organoleptic and physical evaluation criteria described for pharmaceutical and cosmetic formulations (28, 29). Samples with better visual uniformity and lower clumping tendency were selected for the mixing test. 2.3 Mixing test This stage aimed to identify the most efficient mixing method for maximizing the homogeneity of the formulations. Two mixing methods were evaluated, considering that the distribution of MgS within powder blends, as well as its effects on flow-related properties, may depend on blending conditions and the order of component incorporation (26, 27). Method A is described in section 2.2, Figure 2. For Method B (Figure 3), the starch source was first combined with the EO, followed by the addition of MgS and finally SLES, with an intermediate grinding step to enhance dispersion. Only the formulations prepared with MgS were subjected to the mixing test, namely, SLP1-MgS, SLP2-MgS, and SLP3-MgS. At this stage, the EO concentration was reduced to 2%, the dye concentration was halved, and the proportion of SLES was increased. Mixing was carried out in the Incu-Shaker™ Mini at 200 rpm and complemented with manual grinding when necessary to break agglomerates. The formulations were evaluated using the same organoleptic scale described in Table 1. The method that achieved the highest degree of component integration and the best powder flow was selected for preparing prototypes used in subsequent stages of this study. Final prototype selection was based on the overall balance of homogeneity, powder flow, agglomeration tendency, essential-oil incorporation, handling characteristics, and subsequent physical stability. 2.3.1 Score scale To assess component integration and overall formulation uniformity, a qualitative scoring system comprising seven physical and FIGURE 2 Procedure used for mixing the ingredients of the formulations for the homogeneity test. This procedure will be referred to as Method A. The same procedure was applied analogously for formulations containing CA-EO. CA-EO: Cryptocarya alba essential oil. LP-EO: Laureliopsis philippiana essential oil. MgS: Magnesium stearate. SLES: Sodium lauryl ether sulfate.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science05frontiersin.org organoleptic parameters was applied during the homogeneity, mixing, thermal stability, and long-term stability tests. Each parameter was scored on a 1 to 5 scale according to the descriptors presented in Table 1, which were established based on evaluation criteria commonly considered in shampoo and cosmetic formulation studies (24, 28, 30). The evaluations were performed by a single, non-blinded evaluator, corresponding to the researcher conducting experimental procedures. Each sample or experimental condition was assessed once at the corresponding evaluation time point; therefore, no independent replicate organoleptic assessments were performed. Consequently, inter-evaluator concordance and intra-evaluator agreement analyses were not applicable. 2.4 Preliminary physicochemical characterization of the formulations For the macroscopical physicochemical evaluation, cornstarch/ MgS formulations were prepared using mixing method A, resulting in three samples: SLP (LP-carrying formulation), SCA (CA-carrying formulation), and SVE (vehicle formulation). Measurements were performed on 10 g samples. For the present exploratory formulation study, each final prototype (SLP, SCA, and SVE) was prepared as a single experimental batch. Independently prepared replicate batches were not produced; therefore, batch-to-batch reproducibility was not assessed. The tests included pH measurement, thermal TABLE 1 Evaluation criteria for homogeneity, mixing, thermal stability, and long-term stability tests. ScoreVisual homogeneity Absence of lumps Color uniformity Tactile texture FluidityAgglomeration tendency 1 Heterogeneous mixture. Oil is poorly distributed. Big and numerous lumps. Irregularly distributed coloration, presence of clearly defined, colorful, and colorless zones. Extremely rough or compact, hard to handle. The mixture does not flow once the container is inclined. Completely compacted. 2 Slightly heterogeneous. The mixture seems uniform. Dispersed middlesized lumps. Slightly uniform color distribution Saturation differences are scarce. Rough, highly cohesive particles. Slightly fluid Requires strong stirring to move. Persistent lumps. Visibly clumping. 3 Mostly homogeneous. Slightly visible differences in texture. Small lumps. Easily disintegrable. Mostly uniform color. Soft. Lightly rough. The mixture flows slowly without agitation when the container is inclined. Reversible clumping when agitated. 4 Uniform. Non-visible irregular zones. Not visible lumps. Smooth surface. Homogeneous and stable color. Not visible color spots. Fine consistency, homogeneous Smooth to the touch. The mixture flows easily with a slight agitation when the container is inclined. Slight agglomeration. The granular structure is stable. 5 Completely homogeneous, the whole sample is visually uniform. Completely free of lumps. Uniform texture. Completely uniform color intensity is well dispersed throughout the formulation. Silky. Without any palpable roughness. The mixture flows effortlessly without any agitation. No Agglomeration, the mixture stays dispersed. FIGURE 3 Procedure used for mixing the ingredients of the formulations for the mixing test. This procedure will be referred to as Method B. The same procedure was applied analogously for formulations containing CA-EO. CA-EO: Cryptocarya alba essential oil. LP-EO: Laureliopsis philippiana essential oil. MgS: Magnesium stearate. SLES: Sodium lauryl ether sulfate.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science06frontiersin.org stability, mechanical stability, and long-term stability evaluation (28, 29). In addition to the macroscopic stability assessment, the final formulations were subjected to complementary physicochemical characterization. Scanning electron microscopy (SEM) was used to examine particle morphology and surface characteristics. Fouriertransform infrared spectroscopy (FTIR) was performed to compare the spectral profiles of SCA, SLP, and SVE with those of CA-EO and LP-EO, while differential scanning calorimetry (DSC) was used to evaluate the thermal behavior of the formulations and isolated essential oils. Moisture content was determined gravimetrically from the mass loss after heating. Powder flow properties were evaluated through bulk and tapped volume measurements, from which the Hausner ratio (HR) and Carr index (CI) were calculated. HR was calculated as the ratio between tapped density and bulk density, whereas CI was calculated as the relative difference between tapped and bulk density. HR and CI measurements were performed in triplicate for each formulation, while moisture content corresponded to a single determination per formulation. Detailed SEM, FTIR, DSC, moisture, HR, and CI results are provided in the Supplementary Material. pH was determined using rehydrated solutions prepared at a 1:10 ratio (shampoo: distilled water), mixing 0.5 g of sample with 4.5 mL of distilled water. Measurements were performed using a previously calibrated AQUASEARCHER™ AB23PH pH meter to evaluate formulation compatibility and detect potential changes associated with instability or degradation processes (28, 29). Because the formulations were developed as dry powder prototypes, pH adjustment was not performed before the biological evaluations. Modifying the pH of the selected powder system would have required reformulation and subsequent reassessment of homogeneity, powder flow, essential-oil distribution, and physical stability. Therefore, the selected prototypes were evaluated without further pH modification, while the alkaline pH was considered a formulation limitation to be addressed in subsequent optimization stages. Thermal stability was assessed by incubating aliquots of each formulation at 50 °C for 120 h, with organoleptic evaluations conducted every 24 h, while long-term stability was evaluated through bench storage at room temperature for 9 months. In both tests, the organoleptic parameters described in Table 1 were evaluated. Mechanical stability was assessed by centrifugation as an accelerated physical stability test, since centrifugal stress can promote particle rearrangement and reveal tendencies toward compaction, phase separation, sedimentation, or loss of powder uniformity that may not be evident under static storage conditions (29, 31). Briefly, 0.2 g aliquots were centrifuged at 13,000 rpm for 30 min at room temperature to detect signs of compaction, flocculation, or sedimentation. These results were assessed using the qualitative criteria described in Table 2 (28, 29, 31). The thermal, mechanical, and long-term assays were designed primarily to assess macroscopic and physical stability. Quantitative longitudinal chemical profiling of the essential oils during storage was not performed. 2.5 In vitro antibacterial evaluation of the final formulations 2.5.1 Agar diffusion assay The antibacterial activity of the final dry shampoo formulations was evaluated against Staphylococcus pseudintermedius ATCC 49444 and three clinical isolates (SP 1, SP 9, and SP 13). SCA, SLP, and SVE were assessed using an agar diffusion assay, with chlorhexidine 2% included as the reference antiseptic control and an untreated culture as the growth control. Inhibition zone diameters were measured in millimeters after incubation, and assays were performed in triplicate. Complete results are provided in the Supplementary Material. 2.5.2 Serial dilution and minimum inhibitory concentration (MIC) assay The final formulations were additionally evaluated using a serial dilution assay. Before testing, the dry shampoos were dispersed in Mueller–Hinton broth at a 1:3 ratio. SCA and SLP contained 3% of their respective essential oils, and the dilution series was therefore expressed as equivalent essential-oil concentrations. The evaluated concentration range was 30.72 to 0.06 μg/ mL. SVE, which contained no essential oil, was evaluated at the corresponding formulation dilution levels and served as the vehicle control. Positive and negative growth controls were included in each assay. Minimum inhibitory concentration (MIC) was determined as the lowest tested equivalent essential-oil concentration associated with absence of detectable bacterial growth under the evaluated conditions. The complete dilution data are presented in the Supplementary Material. 2.5.3 Viable bacterial counts To provide a quantitative assessment of bacterial survival, aliquots from the serial dilution assay were cultured and viable bacterial TABLE 2 Evaluation criteria for the mechanical stability test. ScoreCompactionFlocculationSedimentation 1 The powder completely compacts; forms a solid and unmanageable mass Large flocs or aggregates are formed; clearly visible clusters that do not disperse The powder separates into distinct layers; high sedimentation is observed 2 Visible compaction of the powder; the mass can be separated after agitation Slight flocculation; dispersed aggregates are present; clusters disperse after agitation Moderate sedimentation, some particles remain suspended 3 No compaction is observed; the powder remains loose No flocculation is observed; the mixture is free of visible aggregates No sedimentation is observed; particles remain fully suspended
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science07frontiersin.org recovery was quantified as colony-forming units per milliliter (CFU/ mL). CFU mL −1 values were calculated as the number of colonies counted × 1,000. These measurements were performed for S. pseudintermedius ATCC 49444 and the clinical isolates SP 1, SP 9, and SP 13. Complete CFU/mL results for each formulation and dilution level are provided in the Supplementary Material. 2.6 Pre-clinical safety test Before exploratory microbiological and tolerability assessment in dogs, a preclinical safety test was performed in mice, comprising skin irritation measurements and an ex vivo histological analysis. For this purpose, eighteen healthy male and female Mus musculus (BALB/c) mice were obtained from the Experimental Platform of the Faculty of Dentistry, University of Chile, in April 2025, to include biological variability. The animals were divided into three groups, with 6 animals each. According to the formulation applied, groups are as follows: group treated with C. alba EO-loaded dry shampoo (SCA), group treated with L. philippiana EO-loaded dry shampoo (SLP) and group treated with the vehicle (SVE). All animals were cared for according to the Ethics Committee of the Odontology Faculty of the University of Chile (CICUA: 24814-ODO-UCH-e3). The subsequent efficacy test in dogs was conducted only after obtaining written informed consent from the owners and approval from the corresponding animal ethics committee. All mice were housed in a conventional facility in a temperature-controlled room with a 12-h light:12-h dark cycle, at the Odontology Faculty of the University of Chile. On June 9th, all animals were weighed, and the hair from a 1 × 2 cm dorsal area was carefully removed using an electric clipper, without the use of chemical depilatory agents. The first formulation application was performed 1 hour after hair removal. Irritation, inflammation, and adverse signs were registered 1, 4, and 8 h after product application. Second doses of the formulations were administered once the 8-h observation period was completed. For days 2, 3, and 4 of the irritation test, the same application protocol was performed. On the fifth day of the irritation test, all animals were euthanized by controlled CO 2 exposure. Immediately after, skin samples of the dorsal treated skin were collected and transferred to 15 mL centrifuge tubes with formaldehyde (10%) for histological analysis (18, 20, 22). 2.6.1 Skin irritation measurements A skin irritation test was conducted to assess the degree of skin irritation associated with shampoo application. Two criteria were used to describe irritability as detailed in Table 3. 2.6.2 Ex-vivo histological analysis For histological evaluation, 5 μm tissue sections were first subjected to deparaffinization with xylene (Biopack) for 15 min. The samples were then rehydrated through sequential ethanol washes of decreasing concentration (100, 95, 80, 70, and 50%), with each step lasting 5 min. Sections were stained with hematoxylin and eosin (H&E, Biopack) for 5 min, followed by removal of excess stain under running water for 2 min. Subsequently, the slides were dehydrated using ascending ethanol concentrations (50, 70, 80, 95, and 100%) and mounted with a microscopy mounting medium (Biopack). After drying, five representative microscopic fields from each experimental group were examined under bright-field microscopy using an Olympus microscope. Images were acquired with a DFC 295 digital camera (Olympus) using a 4 × objective lens and a 10 × ocular lens, corresponding to a total magnification of 40×. Histological sections were independently evaluated by two specialized pathologists who were blinded to the experimental groups. Tissue sections were assessed according to the characteristic histopathological features established for experimental skin injury models, including epithelial integrity, epidermal thickness, epithelial cellularity, inflammatory cell infiltration, blister formation, dermal architecture, edema, vascular congestion, and other morphological alterations associated with tissue injury and repair (32–34). Histopathological findings were interpreted according to these established criteria. In cases of discrepant interpretations, the slides were jointly reviewed until consensus was reached before assigning the final histopathological classification. The histopathological assessment was qualitative, and no numerical semiquantitative grading score was prospectively assigned to the evaluated parameters. 2.7 Microbiological evaluation in dogs 2.7.1 Study design, ethical approval, randomization, allocation concealment, and blinding An exploratory, prospective, randomized, controlled, four-arm parallel-group clinical study was conducted in client-owned dogs clinically diagnosed with superficial bacterial pyoderma. Dogs were recruited from several veterinary care centers in Santiago, Chile, including private clinics and university-affiliated veterinary services. The study protocol was approved by the corresponding animal ethics committee under approval code ID14/2024, dated 28 June 2024. Written informed consent was obtained from all owners before enrollment. TABLE 3 Score system used to evaluate irritation. ScoreErythemaEdema 0Without lesionWithout edema 1Slightly reddenedSlight edema 2Moderately reddenedModerately sized edema (>1 mm elevation) 3Severely reddened or burnt>1 mm elevation and extended beyond treated area
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science08frontiersin.org Thirty-two dogs were allocated in a 1:1:1:1 ratio to four parallel treatment groups, with eight dogs planned per group: shampoo containing Cryptocarya alba essential oil (SCA), shampoo containing Laureliopsis philippiana essential oil (SLP), vehicle shampoo (SVE), or the chlorhexidine-based commercial comparator Inveclor ® . During the study, oclacitinib or comparable non-corticosteroid antipruritic therapies were permitted when clinically required. In contrast, corticosteroids, systemic or topical antibiotics, antiseptics, and natural products with potential antimicrobial or anti-inflammatory activity were not allowed. Dogs were not eligible for enrollment if they had received systemic or topical antibiotics, corticosteroids of any class, or natural products with potential antimicrobial or anti-inflammatory activity within the 15 days preceding baseline evaluation. Therefore, all enrolled dogs completed a minimum 15-day treatment-free period before microbiological sampling and initiation of the assigned topical treatment. The random allocation sequence was generated using the Datacapt Randomization platform. Simple randomization was applied at the individual-dog level, without stratification, blocking, or other restrictions. The randomization sequence and the correspondence between study codes and treatment groups were accessible only to Daniela Siel, who served as the allocation custodian and was not involved in the blinded clinical outcome assessments, clinical laboratory analyses, or microbiological evaluations. Allocation concealment was maintained by restricting access to the randomization sequence and treatment-code list to the allocation custodian. Eligibility was confirmed, written owner consent was obtained, and each dog was enrolled and assigned an individual study identification code before the corresponding treatment allocation was accessed. The clinicians responsible for recruitment and clinical evaluation had no access to the allocation sequence and could not predict the treatment assigned to the next enrolled dog. All study products were dispensed in identical containers labeled exclusively with the corresponding study identification code. The containers did not display the formulation name, essential oil, vehicle, or comparator identity. Although all products were provided in identical coded containers, the effectiveness of blinding was not formally assessed, and treatment identification based on potential organoleptic differences cannot be completely excluded. Dog owners, treating clinicians, and clinicians responsible for follow-up and outcome assessment were blinded to treatment allocation throughout the study. Clinical records and biological samples were identified exclusively using coded study identification numbers. Personnel performing hematological and biochemical analyses, cytological assessments, bacterial culture, colony-count evaluation, and bacterial identification using the VITEK ® system were also blinded to treatment allocation. Treatment codes were not disclosed to clinical or laboratory assessors until completion of the corresponding evaluations and analyses. Reporting of the randomization, allocation-concealment, implementation, and blinding procedures was structured according to the PetSORT recommendations for randomized trials in client-owned dogs and cats (35). The primary exploratory outcome of the canine study was the microbiological response at Day 21, assessed through bacterial culture, bacterial detection, and predefined bacterial-burden categories based on CFU counts. Clinical observations performed during followup were considered supportive assessments of local tolerability and general clinical evolution rather than independent quantitative efficacy outcomes. Because a standardized quantitative lesion-severity score was not prospectively applied and complete standardized photographic records were not available for all enrolled dogs, no retrospective clinical lesion score was generated. Following allocation, the assigned topical product was applied by the owner directly to the affected skin areas twice daily, ideally at approximately 12-h intervals, for 21 consecutive days. The product was applied using protected hands and was left on the skin without rinsing. After each application, owners were instructed to prevent the dogs from licking, grooming, or otherwise removing the product for approximately 10–20 min. Standardized instructions were provided to the owners to promote consistent product administration. Clinical follow-up was conducted on Days 0, 7, 14, and 21. 2.7.2 Microbiological sampling and bacterial identification Microbiological specimens were collected from dogs clinically diagnosed with superficial bacterial pyoderma before treatment initiation and after completion of the 21-day topical treatment period. Samples were taken from active cutaneous lesions using sterile cotton swabs, according to standard approaches for microbiological evaluation in canine superficial pyoderma (17, 36). Prior to sampling, superficial debris was carefully removed from the lesion area with sterile gauze, without applying antiseptic agents, in order to avoid affecting bacterial recovery. The swab was gently rolled across the lesion surface to collect exudate and bacterial material, and samples were promptly transported under refrigerated conditions to the MICROVET Diagnostic Laboratory, Faculty of Veterinary and Animal Sciences, University of Chile, for further processing. Upon arrival, samples were inoculated onto blood agar plates and incubated at 37 °C for 24–48 h. Colonies with morphological characteristics suggestive of staphylococci were selected and subcultured to obtain pure bacterial isolates. Species identification was then carried out using the VITEK ® automated identification system (bioMérieux), enabling identification of Staphylococcus pseudintermedius at the species level (19). For each sample, the presence or absence of S. pseudintermedius was recorded. Detection of other Staphylococcus spp. was also considered to further describe the bacterial profile of the lesions. Isolates were classified as S. pseudintermedius, Staphylococcus aureus, or other Staphylococcus species. Antimicrobial susceptibility was evaluated using the corresponding automated susceptibility panel. Oxacillin susceptibility was used as the phenotypic indicator for the identification of methicillin-resistant S. pseudintermedius (MRSP), with isolates showing resistance to oxacillin considered phenotypically compatible with MRSP. To assess the microbiological response to treatment, bacterial burden was estimated by counting colony-forming units (CFU) recovered from lesion swab cultures at baseline and after 21 days of topical dry shampoo application. Baseline cultures were considered positive when bacterial counts exceeded 300 CFU/swab, corresponding to a very high or uncountable bacterial load. After treatment, bacterial growth was categorized as absent or non-significant when 0–15 CFU/ swab were detected, countable when values ranged from 16 to
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science09frontiersin.org 300 CFU/swab, and uncountable when bacterial counts exceeded 300 CFU/swab. 2.8 Sample-size calculation An a priori sample-size calculation was performed using G*Power version 3.1.9.6 and independently verified using R version 4.4.3 within RStudio version 2024.12.1. The expected effect size was estimated from unpublished preliminary data generated by our research group. For the overall comparison of the microbiological response among four equally allocated treatment groups, the calculation considered Cohen’s w = 0.60, a two-sided type I error rate of 0.05, and 80% statistical power. The analysis indicated that a minimum total sample size of 32 dogs was required, corresponding to eight animals per group. This sample size was also consistent with VICH GL43 recommendations applicable to target-animal safety and local tolerance evaluations of dermally administered veterinary products. The study was designed to detect large microbiological effects and common tolerability concerns and was not powered to detect small or moderate effects or to establish equivalence or non-inferiority among treatments. 2.9 Statistical analysis Statistical analyses were performed using GraphPad Prism version 10 and R version 4.4.3 within RStudio version 2024.12.1. The statistical approach and revised analyses were reviewed in consultation with a biostatistician. The individual animal was considered the experimental unit. All statistical tests were two-sided, and statistical significance was established at p < 0.05. pH measurements were technical replicates and were therefore summarized descriptively as mean ± standard deviation without inferential statistical comparison. For continuous outcomes analyzed using parametric methods, model assumptions were evaluated primarily through graphical inspection of residuals, including quantile–quantile plots and residual-versus-fitted plots. Normality of model residuals was additionally assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using the Brown–Forsythe test. Because formal normality tests have limited power in small samples, selection of the statistical approach was based on the combined assessment of graphical diagnostics, residual distribution, and variance homogeneity rather than on the Shapiro–Wilk test alone. When parametric assumptions were not adequately satisfied, appropriate non-parametric or robust alternatives were used. Microbiological outcomes were analyzed according to their scale and data structure. Bacterial detection at Day 21 was treated as a binary categorical outcome, whereas bacterial burden was analyzed according to the predefined CFU categories. Because of the limited sample size and sparse cell counts, overall between-group comparisons at Day 21 were performed using the Fisher–Freeman–Halton exact test. Cramér’s V was calculated as an effect-size measure for the overall association between treatment group and categorical microbiological outcomes. Baseline-to-Day-21 changes in bacterial detection were evaluated using the exact McNemar test, accounting for the paired nature of repeated observations within the same dog. Only animals with microbiological results available at both time points were included in paired analyses. Changes in bacterial species identification were summarized descriptively using individual transition frequencies because several categories contained sparse or zero cell counts, precluding reliable multicategorical paired inference. Missing observations were not imputed, and the number of animals contributing to each analysis is reported. Where secondary pairwise comparisons were performed, p-values were adjusted using the Holm procedure. For multiple hematological and biochemical safety outcomes, the Benjamini–Hochberg procedure was used to control the false discovery rate. Both unadjusted and adjusted p-values were reported where relevant, with statistical inference based on adjusted values when multiplicity correction was applicable. Missing observations were not imputed, and the number of animals contributing to each analysis is reported. 3 Results 3.1 Homogeneity test Organoleptic parameters—visual appearance, absence of lumps, color consistency, tactile texture, fluidity, agglomeration tendency, and aroma intensity (Table 1)—were used to assess formulation uniformity and handling properties, which are key factors for the performance and acceptability of topical powders. As shown in Table 4, formulations containing MgS obtained higher general mean scores than their counterparts without MgS in the cornstarch- and tapioca-based matrices. The general mean score increased from 2.6 in SLP1 to 4.0 in SLP1-MgS, and from 3.4 in SLP2 to 4.7 in SLP2-MgS. The mixed-starch TABLE 4 Organoleptic scores obtained for all formulations evaluated in the homogeneity test. ParameterSLP1SLP1-MgSSLP2SLP2-MgSSLP3SLP3-MgS Visual homogeneity2.04.03.05.02.04.0 Absence of lumps2.03.03.04.02.04.0 Color uniformity2.05.03.05.03.05.0 Tactile texture2.03.04.05.03.04.0 Fluidity3.04.03.04.03.04.0 Agglomeration tendency2.04.03.05.03.04.0 Aroma intensity5.05.05.05.05.05.0 General mean2.64.03.44.73.04.3 SLP, Shampoo with Laureliopsis philippiana essential oil.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science10frontiersin.org formulation also showed an increase in the general mean score after MgS incorporation, from 3.0 in SLP3 to 4.3 in SLP3-MgS. Among all formulations evaluated, SLP2-MgS showed the highest overall performance, with a general mean score of 4.7, followed by SLP3-MgS and SLP1-MgS, with mean scores of 4.3 and 4.0, respectively. Aroma intensity remained constant across all formulations, with a score of 5. Overall, the tapioca-based formulation containing MgS showed the highest mean score (4.7), compared with 2.6, 4.0, 3.4, 3.0, and 4.3 for SLP1, SLP1-MgS, SLP2, SLP3, and SLP3-MgS, respectively. Formulations containing MgS also exhibited lower agglomeration tendency and improved EO dispersion within the matrix. 3.2 Mixing test Mixing test results are summarized in Table 5. Overall, Method A produced mixtures (SLP1-MA, SLP2-MA, and SLP3-MA) with improved flow and lower particle cohesion, while Method B (SLP1-MB, SLP2-MB, and SLP3-MB) generated fine lumps and greater agglomeration, indicating less efficient dispersion of both the solid matrix and the EO. Color uniformity did not vary between formulations and mixing methods, indicating that both methods produced an equally distributed EO in the formulation. The lump formation tendency score was lower for SLP3-MgS than for SLP1-MgS and SLP2-MgS formulations, and independent of the mixing method used. 3.3 Macroscopic physicochemical characterization of the optimized formulation 3.3.1 pH measurements pH measurements (Table 6) showed slight variation among SLP, SCA, and SVE. All three formulations exhibited similar alkaline values, ranging from 10.93 to 11.16, with low variability, as reflected by the small standard deviations. These results indicate that incorporation of the essential oils did not markedly alter the pH of the powder matrix. Given the alkaline nature of the formulations, their suitability for repeated topical application and potential implications for dermal tolerability are discussed further in Section 4.2.1. 3.3.2 Thermal stability Thermal stability results (Tables 7–9) showed gradual changes in sensory and physical properties with increasing incubation time. Visual homogeneity and color uniformity remained unchanged throughout the 120-h assay, whereas tactile texture, fluidity, TABLE 5 Organoleptic scores obtained for the formulations evaluated using mixing Method A and Method B in the mixing test. ParameterSLP1-MASLP1-MBSLP2-MASLP2-MBSLP3-MASLP3-MB Visual homogeneity5.04.04.03.04.03.0 Absence of lumps4.03.03.03.03.03.0 Color uniformity5.05.05.05.05.05.0 Tactile texture4.04.04.03.04.04.0 Fluidity5.05.04.04.03.02.0 Agglomeration tendency4.03.04.03.02.02.0 Aroma intensity3.03.03.03.03.03.0 General mean4.33.93.93.43.43.1 SLP, shampoo with Laureliopsis philippiana essential oil, prepared through Method A and B (MA, MB). TABLE 6 pH values for all formulations. FormulationpH SLP10.93 ± 0.06 SCA11.01 ± 0.03 SVE11.16 ± 0.03 SLP, shampoo containing Laureliopsis philippiana essential oil; SCA, shampoo containing Cryptocarya alba essential oil; SVE, vehicle shampoo. TABLE 7 Thermal stability score for shampoo loaded with Laureliopsis philippiana essential oil (SLP). Parameter024487296120 Visual homogeneity 4.04.04.04.04.04.0 Absence of lumps3.53.03.03.03.03.0 Color uniformity4.04.04.04.04.04.0 Tactile texture4.03.03.02.02.02.0 Fluidity4.04.04.03.53.03.0 Agglomeration tendency 3.53.03.03.02.02.0 Aroma intensity4.02.02.02.02.02.0 General mean3.93.33.33.12.92.9 TABLE 8 Thermal stability score for shampoo loaded with Cryptocarya alba essential oil (SCA). Parameter024487296120 Visual homogeneity 4.0 4.04.04.04.04.0 Absence of lumps3.53.02.52.52.03.0 Color uniformity4.04.04.04.04.04.0 Tactile texture4.04.03.02.52.52.5 Fluidity4.04.04.03.73.03.0 Agglomeration tendency 3.5 3.03.03.02.02.0 Aroma intensity3.02.52.02.02.02.0 General mean3.83.53.23.02.82.8
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science11frontiersin.org agglomeration tendency, and aroma intensity showed progressive decreases in score. Both EO-containing formulations exhibited a reduction in aroma intensity during thermal exposure, consistent with loss of volatile components; however, the chemical composition of the essential oils was not quantitatively assessed during this assay. Therefore, these findings are interpreted as evidence of preliminary physical stability under the evaluated thermal conditions rather than confirmation of chemical stability. 3.3.3 Mechanical stability Centrifugation data (Table 10) shows that all formulations obtained a compaction score of 2. For flocculation, SLP and SCA obtained a score of 2, whereas SVE showed the highest score, with a value of 3. For sedimentation, SLP and SVE obtained scores of 3, while SCA showed a lower score of 2. The general mean scores were 2.3 for SLP, 2 for SCA, and 2.7 for SVE, with SVE showing the highest general mean score in the mechanical stability test, followed by SLP and SCA. 3.3.4 Long-term stability Long-term stability test results (Tables 11–13) showed preservation of several macroscopic properties of the three formulations tested (SLP, SCA, and SVE) during the 9-month evaluation period. Visual homogeneity and color uniformity remained unchanged throughout the 9-month evaluation period in all formulations. For SLP, the general mean score decreased from 3.9 at baseline to 3.0 at 3 months, and to 2.9 at both 6 and 9 months. For SCA, the general mean score decreased from 3.7 at baseline to 2.9 at 3 and 6 months, and to 2.7 at 9 months. For SVE, the general mean score decreased from 4.0 at baseline to 3.6 at 3 months, and to 3.4 at both 6 and 9 months. Aroma intensity decreased from 5 to 3 after 3 months in both EO-containing formulations and remained at this score until month 9, whereas SVE remained odorless throughout the assay. Scores related to tactile texture, fluidity, and agglomeration tendency showed reductions over time, while absence of lumps remained constant in SVE and decreased after 3 months in both EO-containing formulations. Overall, visual homogeneity and color uniformity were maintained during the nine-month storage period, while gradual changes were observed in texture, fluidity, agglomeration tendency, and aroma intensity. These results indicate preservation of the macroscopic appearance of the formulations with progressive changes in powder-handling properties over time. Because no quantitative chemical analysis of the essential-oil composition TABLE 9 Thermal stability score for shampoo for SVE. Parameter024487296120 Visual homogeneity4.04.04.04.04.04.0 Absence of lumps4.03.03.03.02.52.0 Color uniformity5.05.05.05.05.05.0 Tactile texture4.04.04.03.53.02.5 Fluidity4.04.04.04.03.53.0 Agglomeration tendency 3.0 3.03.03.02.52.5 Aroma intensity0.00.00.00.00.00.0 General mean3.43.33.33.22.92.7 TABLE 10 Score for shampoo loaded with SLP, SCA, and SVE. ParameterSLPSCASVE Compaction2.02.02.0 Flocculation2.02.03.0 Sedimentation3.02.03.0 General mean2.32.02.7 TABLE 11 Long-term stability score for shampoo loaded with Laureliopsis philippiana essential oil (SLP). Parameter0 m3 m6 m9 m Visual homogeneity4.04.04.04.0 Absence of lumps3.02.02.02.0 Color uniformity4.04.04.04.0 Tactile texture3.02.02.02.0 Fluidity4.03.02.02.0 Agglomeration tendency 4.03.0 3.0 3.0 Aroma intensity5.03.03.03.0 General mean3.93.02.92.9 TABLE 12 Score for SCA. Parameter0 m3 m6 m9 m Visual homogeneity4.04.04.04.0 Absence of lumps3.02.02.02.0 Color uniformity4.04.04.04.0 Tactile texture3.02.02.02.0 Fluidity4.03.03.02.0 Agglomeration tendency 3.02.02.02.0 Aroma intensity5.03.03.03.0 General mean3.72.92.92.7 TABLE 13 Score for SVE. Parameter0 m3 m6 m9 m Visual homogeneity4.04.04.04.0 Absence of lumps4.04.04.04.0 Color uniformity5.05.05.05.0 Tactile texture5.04.04.04.0 Fluidity5.04.04.04.0 Agglomeration tendency 5.04.0 3.0 3.0 Aroma intensity0.00.00.00.0 General mean4.03.63.43.4
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science12frontiersin.org was performed during storage, these observations should be interpreted as preliminary evidence of physical rather than chemical stability. 3.3.5 Complementary physicochemical characterization Additional physicochemical characterization of the final dry shampoo formulations was performed by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), moisture determination, and powder flow measurements (Supplementary Figures S1–S3 and Supplementary Table S4). SEM examination showed a heterogeneous particulate morphology in SCA, SLP, and SVE, with granular structures and particle aggregates observed throughout the samples (Supplementary Figure S1). FTIR analysis showed broadly similar spectral profiles among SCA, SLP, and SVE, with the formulation spectra predominantly characterized by the bands associated with the common powder matrix (Supplementary Figure S2). The isolated CA-EO and LP-EO exhibited distinct spectral features, particularly within the fingerprint region, whereas no major new absorption bands were observed in the EO-containing formulations compared with the vehicle formulation. DSC thermograms showed a similar overall thermal profile for the three dry shampoo formulations over the evaluated temperature range (Supplementary Figure S3). SCA and SLP displayed thermal events comparable to those observed in SVE, while the isolated essential oils showed comparatively limited thermal transitions within the analyzed range. No marked additional thermal event attributable exclusively to EO incorporation was evident in the formulated powders. Powder flow measurements showed Hausner ratios of 1.62 ± 0.02, 1.63 ± 0.08, and 1.70 ± 0.06 for SCA, SLP, and SVE, respectively. The corresponding Carr index values were 38.30 ± 0.74%, 38.39 ± 3.09%, and 40.92 ± 3.47%, respectively (Supplementary Table S4). Moisture contents were 12.61% for SCA, 14.02% for SLP, and 10.69% for SVE. Overall, SCA and SLP showed similar flow-related parameters, whereas SVE presented the highest Hausner ratio and Carr index. 3.4 Preliminary antimicrobial activity As an initial assessment of the antimicrobial activity of the dry shampoo formulations, agar diffusion assays were performed against selected clinical isolates of S. pseudintermedius (Supplementary Table S5). Neither SCA nor SLP produced measurable inhibition zones against the three tested clinical isolates, whereas the 2% chlorhexidine control showed clear inhibition, with mean inhibition zones ranging from 19.73 to 23.85 mm. These findings indicate that the dry shampoo formulations did not exhibit detectable antimicrobial activity under the conditions of the agar diffusion assay. As a complementary exploratory assessment, the antimicrobial activity of the dry shampoo formulations was evaluated by broth microdilution against selected S. pseudintermedius strains (Supplementary Table S6), together with quantitative viable bacterial counts across the corresponding dilution levels (Supplementary Tables S7–S10). In contrast to the agar diffusion results, SCA and SLP showed inhibitory activity at equivalent EO concentrations ranging from 0.12 to 0.24 μg/mL across the tested strains. The viable-count data showed lower bacterial recovery at higher formulation concentrations and increasing CFU/mL as the formulations were progressively diluted (Supplementary Tables S7–S10). However, inhibitory responses and reduced bacterial recovery were also observed with the vehicle formulation (SVE), indicating that the observed response was not restricted to the EO-containing formulations. 3.5 Irritation results All measurements of skin irritation were scored at 0, showing that neither the EO-containing formulations nor the vehicle produced erythema or edema at the treated site. Serial photographic documentation (Figure 4) enabled evaluation of the cutaneous FIGURE 4 Photographic record of the shaved dorsal area of three mice (first row: Prototype containing LP-EO; second row: Prototype containing CA-EO; third row: Vehicle without EO during the safety assay), showing no evident macroscopic skin alterations.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science13frontiersin.org response in the shaved and demarcated dorsal area corresponding to the site of formulation application. Throughout the observation period, the animals’ skin remained intact, with no macroscopic signs of cutaneous irritation, including erythema, edema, ulceration, exudation, severe desquamation, open lesions, crust formation, bleeding, or necrotic areas. The treated area maintained a regular boundary and homogeneous coloration over time. General welfare parameters—including appearance, food and water intake, body temperature, respiratory and heart rate, and spontaneous and provoked behavior—remained within normal ranges throughout the experiment. Body weight showed minor fluctuations during the study, including both increases and decreases generally below 5%, with no consistent pattern across individuals, cages, or treatments. No progressive or sustained weight loss was observed during the follow-up period. 3.6 Histological analysis Tissue samples from the dorsal skin of mice were collected to evaluate, at the cellular level, potential signs of irritation, inflammation, or tissue damage associated with the application of the formulations. Histological analysis (Figure 5) revealed that the cutaneous architecture appeared preserved, with a continuous, well-defined epidermis and no evidence of ulceration, erosion, or marked hyperplasia. The underlying dermis maintained a normal organization of collagen fibers, without apparent inflammatory infiltrates or signs of interstitial edema. Likewise, visible skin appendages exhibited preserved morphology, consistent with healthy tissue. Taken together, the comparative histological analysis demonstrates that none of the evaluated formulations induced evident histopathological alterations in the animal’s skin, with findings consistent with physiologically normal tissue. 3.7 Microbiological evaluation in dogs To provide preliminary evidence of antimicrobial activity in the target species, a microbiological evaluation was conducted in clientowned dogs diagnosed with superficial pyoderma. Dogs were initially allocated into four treatment groups. However, one dog from the vehicle shampoo group discontinued the study before the final microbiological evaluation; therefore, the final microbiological analysis included eight dogs in the SCA group, eight dogs in the SLP group, seven dogs in the SVE group, and eight dogs in the Inveclor ® group. At baseline, all but one dog were culture-positive for Staphylococcus spp., with S. pseudintermedius being the predominant species identified. On Day 21, S. pseudintermedius remained the most frequently detected species; however, changes in bacterial identification were observed in some animals, including transitions between S. FIGURE 5 Histological samples obtained from mouse epidermis, grouped by shampoo formulation. Images were acquired by light microscopy using a 4 × objective lens and a 10 × ocular lens, corresponding to a total magnification of 40×. SLP: Shampoo with LP-EO, SCA: Shampoo with CA-EO, SVE: Vehicle shampoo without EO. TABLE 14 Bacterial strains identified by VITEK® between day 1 and day 21 in dogs with superficial pyoderma (n = 31). Strain identified on day 1 Strain identified on day 21 S. pseudintermediusS. aureusOther spp.No bacteria S. pseudintermedius15528 S. aureus0000 Other spp.1000 The data represents the number of dogs showing each transition in bacterial species between baseline (day 1) and at the end of treatment (day 21). “Other spp.” includes Aerococcus viridans and Staphylococcus warneri. “No bacteria” indicates absence of detectable cocci in culture on day 21.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science14frontiersin.org pseudintermedius and S. aureus, shifts toward other staphylococcal species, and cases in which no cocci were detected in culture (Table 14). Several transitions were observed, including persistence of S. pseudintermedius, transitions to other staphylococcal species, and absence of detectable cocci at Day 21. Because several categories contained sparse or zero cell counts, no formal inferential comparison of multicategorical species transitions was performed. At Day 21, bacterial elimination, defined as the absence of detectable cocci in culture, was achieved in the SCA and Inveclor ® groups, but not in the SVE and SLP groups. The distribution of bacterial-load categories at Day 21 differed numerically among treatment groups but did not reach statistical significance (Fisher–Freeman–Halton exact test, p = 0.639; Cramér’s V = 0.277). The highest bacterial elimination rate was observed in the Inveclor ® group, with absence of detectable cocci in 4 of 8 dogs (50.0%). Among the EO-loaded shampoo groups, bacterial elimination was observed only in the SCA group, with 3 of 8 dogs (37.5%) showing absence of detectable cocci at Day 21. In contrast, all dogs in the SLP group remained culture-positive, as did all dogs in the vehicle shampoo group (SVE), with bacterial presence detected in 8 of 8 and 7 of 7 dogs, respectively (Table 15). Although the effect-size estimate suggested a non-negligible association between treatment group and bacterial-load category, the small group sizes and sparse cell counts resulted in considerable uncertainty. These findings should therefore be interpreted as exploratory. Additionally, bacterial load was categorized at Day 21 to provide a more detailed assessment of the microbiological response. The highest proportion of dogs reaching the non-significant bacterial load category was observed in the SCA group (4/8; 50.0%), followed by the Inveclor ® group (3/8; 37.5%), the SVE group (2/7; 28.6%), and the SLP group (1/8; 12.5%). Countable bacterial loads were observed in 2 of 8 dogs from the SCA group (25.0%), 1 of 8 dogs from the SLP group (12.5%), 2 of 7 dogs from the SVE group (28.6%), and 2 of 8 dogs from the Inveclor ® group (25.0%). The highest proportion of dogs remaining in the uncountable category was observed in the SLP group (6/8; 75.0%), followed by the SVE group (3/7; 42.9%), the Inveclor ® group (3/8; 37.5%), and the SCA group (2/8; 25.0%; Table 16). Comparison of bacterial load distribution among treatment groups using the Fisher–Freeman–Halton exact test did not reveal statistically significant differences (p = 0.8781). At Day 21, bacterial growth was categorized as non-significant or significant to assess the microbiological response among treatment groups (Figure 6). The highest proportion of dogs with non-significant bacterial growth was observed in the SCA group, with 4 of 8 dogs (50.0%), followed by the Inveclor ® group, with 3 of 8 dogs (37.5%). In the SVE group, non-significant growth was observed in 2 dogs, while 5 showed significant growth and 1 had no available information. In the case of the SLP group, one dog showed non-significant bacterial growth at Day 21, while significant bacterial growth was observed in the remaining cases. When bacterial growth was dichotomized according to the predefined CFU threshold, no statistically significant overall difference was detected among treatment groups (Fisher–Freeman–Halton exact test (p = 0.485); Cramér’s V = 0.2). SCA showed the highest proportion of dogs reaching the lower bacterial-growth category. However, confidence intervals were wide and the estimated between-group differences remained imprecise. Antimicrobial susceptibility testing showed that none of the S. pseudintermedius isolates recovered during the study were resistant to oxacillin. Therefore, no isolates phenotypically compatible with MRSP were detected. 4 Discussion 4.1 Shampoo formulation 4.1.1 Homogeneity test MgS is an established excipient in pharmaceutical and cosmetic powder systems, commonly used as a lubricant and anti-adherent to improve flow by reducing particle–particle friction and cohesion (26, 27, 37). These properties were considered relevant for the present TABLE 15 Bacterial presence and absence on day 21 by treatment group in dogs with superficial pyoderma. GroupnPresenceAbsence SLP880 (0.0%) SCA853 (37.5%) SVE770 (0.0%) Inveclor®844 (50%) Presence indicates detection of any Staphylococcus species by culture and VITEK® identification on day 21. Absence indicates no detectable cocci in culture. SCA, shampoo containing Cryptocarya alba essential oil; SLP, shampoo containing Laureliopsis philippiana essential oil; SVE, vehicle shampoo. TABLE 16 Bacterial load categorization on day 21 by treatment group. Bacterial load was categorized based on CFU counts on day 21. Non-significant: 0–15 CFU/swab; Countable: 16–300 CFU/swab; Uncountable: >300 CFU/swab. GroupnNonsignificant CountableUncountable SLP81 (12.5%)1 (12.5%)6 (75.0%) SCA84 (50.0%)2 (25.0%)2 (25.0%) SVE72 (28.6%)2 (28.6%)3 (42.9%) Inveclor®83 (37.5%)2 (25.0%)3 (37.5%) FIGURE 6 Significant growth (>15 CFU) or non-significant growth of cocci in final microbiological cultures (Day 21) across treatment groups. Fisher–Freeman–Halton exact test did not reveal significant differences among treatment groups (p = 0.485). Gray indicates unavailable data. SLP: Shampoo containing L. philippiana essential oil; SCA: Shampoo containing C. alba essential oil; SVE: Vehicle shampoo without antimicrobial agent.
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science15frontiersin.org starch-based formulations because the incorporation of a lipophilic EO may promote agglomeration, uneven distribution, and increased cohesion within the powder matrix. Consistently, formulations containing MgS (SLP1-MgS, SLP2-MgS, and SLP3-MgS) showed greater visual uniformity, better flow, and lower agglomeration tendency than their MgS-free counterparts (Table 4). These properties may favor more uniform incorporation of the EO within the powder matrix and more consistent handling during topical application (10, 38). The similar aromatic intensity observed among formulations indicates comparable organoleptic aroma intensity at the initial evaluation. Overall, these findings support the inclusion of MgS in the selected formulation for subsequent physicochemical characterization and stability testing (31, 39). Although other flow-enhancing excipients may also be suitable, this preliminary optimization stage was intended to evaluate the functional contribution of MgS rather than to perform an exhaustive screening of glidants or anti-caking agents. 4.1.2 Mixing test Efficient mixing is particularly important in powder systems containing volatile lipophilic compounds, as adequate dispersion contributes to formulation uniformity and more consistent distribution of the active components within the powder matrix. Comparing both mixing methods (Figures 2, 3), one of the key differences is the sequential order of mixing the starch source, the EO, and the MgS. Given the fact that MgS acts as an antiagglomerant, the initial mixing step of MgS and the starch source in mixing method A resulted in overall better performance in visual homogeneity and agglomeration tendency (Table 5) than method B. Aromatic intensity remained similar between samples prepared with both procedures, indicating that no marked organoleptic difference in aroma intensity was detected between the two mixing methods (Table 5) (38, 39). When considering the combined results of the homogeneity and mixing evaluations, the cornstarch-based formulation containing MgS and prepared using Method A was selected as the base prototype for subsequent studies. Although SLP2-MgS obtained the highest individual mean score in the initial homogeneity test, the cornstarchbased formulation showed the most favorable overall balance of visual uniformity, flow behavior, agglomeration tendency, and performance under the selected mixing procedure. An additional limitation concerns the preliminary nature of the formulation optimization process. Organoleptic parameters were evaluated by a single non-blinded evaluator, with each formulation assessed once at each corresponding time point; therefore, independent replicate assessments and inter- or intra-observer agreement analyses were not performed, potentially introducing observer-dependent bias into the selection of the optimized formulations. In addition, each final prototype was prepared as a single experimental batch, precluding assessment of batch-to-batch reproducibility. Although the complementary quantitative and analytical characterization incorporated in this study, including moisture content, Hausner ratio, Carr index, SEM, FTIR, and DSC analyses, provides additional objective information on the technological and physicochemical properties of the selected formulations (29, 31), these measurements do not eliminate the limitations associated with the initial qualitative screening process or the lack of independent batch replication. Future formulation-development studies should therefore incorporate replicated quantitative measurements, blinded assessment by multiple evaluators, objective powder-characterization parameters from the early stages of formulation selection, and independently prepared batches to confirm the reproducibility of physicochemical characteristics, powder-flow behavior, EO distribution, and stability across manufacturing runs. 4.2 Physicochemical characterization and stability 4.2.1 pH The alkaline values obtained are consistent with the nature of powder-based formulations intended for dry cleansing, where the presence of absorbent excipients may generate slightly basic environments. Similar behavior has been described in solid matrices incorporating EOs (10, 38). The comparable pH values observed for SLP, SCA, and SVE indicate that EO incorporation did not markedly alter this parameter. However, all formulations exhibited alkaline pH values close to 11, which are above the ranges most commonly reported for canine skin. Canine skin pH is generally reported between 5.5 and 7.2, although broader ranges from 4.84 to 9.95 have also been described (40). In addition, commercially available non-medicated canine shampoos have been reported to exhibit pH values ranging from 5.7 ± 0.09 to 8.4 ± 0.18 (41). This difference is relevant because prolonged exposure to highly alkaline conditions may affect epidermal barrier function and potentially increase susceptibility to irritation, particularly in inflamed or compromised skin. In the present study, pH was measured after rehydration of the dry powder at a 1:10 shampoo:water ratio and therefore does not necessarily represent the exact microenvironment generated by the dry formulation after topical application. Nevertheless, the alkaline values remain an important limitation of the current prototypes, particularly considering the reported physiological pH range of canine skin and the pH values described for commercially available canine shampoos (40, 41). pH adjustment was not performed before the biological studies because the selected formulations had already been optimized as dry powder systems, and modification of this parameter would have required reformulation followed by reassessment of homogeneity, powder flow, EO distribution, and physical stability (28, 29, 31). Although no erythema, edema, or histological alterations were observed in the murine safety assay under the evaluated conditions, these findings do not exclude possible effects associated with repeated or prolonged application to canine skin. This consideration is relevant because topical tolerability may depend on exposure conditions and skin-barrier status, particularly in compromised or inflamed skin (18, 40). Therefore, future formulation development should include pH optimization followed by renewed physicochemical characterization and safety evaluation in the target species before extended clinical use. 4.2.2 Thermal stability test Thermal stability results (Tables 7–9) revealed gradual changes in sensory and physical properties with increasing incubation time. Visual attributes such as uniformity and color remained unchanged throughout the assay, indicating preservation of the macroscopic appearance of the formulations under the evaluated thermal conditions. Both EO-containing formulations (SLP and SCA) exhibited similar behavior, with slight decreases in powder flow and tactile texture over time, accompanied by a
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science16frontiersin.org gradual increase in agglomeration tendency and a reduction in aromatic intensity, which is compatible with partial loss of volatile EO constituents. The essential oils incorporated into the formulations contain several volatile constituents. As previously characterized, LP-EO was predominantly composed of linalool, eucalyptol, and safrole, whereas CA-EO showed a more complex profile, with sabinene and eucalyptol among its major constituents (23). The volatile nature of these compounds may contribute to changes in aroma intensity during exposure to elevated temperatures (38). The preservation of visual parameters suggests that no major macroscopic instability occurred during thermal exposure, whereas the progressive changes in texture, agglomeration tendency, and flow indicate alterations in the physical behavior of the powders over time. During thermal incubation, SVE showed gradual reductions in fluidity and absence-of-lumps scores, indicating changes in powder handling properties over time. Overall, the results suggest that the EO-containing formulations maintained macroscopic stability broadly comparable to the vehicle, with the main changes limited to gradual variations in texture and aroma under elevated temperature conditions (31, 38). Importantly, the thermal assay primarily assessed changes in macroscopic and physical properties. Although the decrease in aroma intensity is compatible with the loss of volatile components, the chemical composition of the essential oils was not quantitatively monitored during thermal exposure. Essential oils are complex mixtures whose constituents may undergo volatilization, oxidation, and chemical degradation depending on temperature and storage conditions (38). Therefore, oxidation, degradation, or selective volatilization of individual EO constituents cannot be excluded, and the present data should not be interpreted as demonstrating complete chemical stability. Quantitative longitudinal analysis, such as GC–MS profiling before and after thermal exposure, would be required to confirm the stability of individual EO constituents (31). 4.2.3 Centrifugation stability test The centrifugation stability test (Table 10) showed that all formulations presented the same degree of compaction after centrifugation. Differences were observed in flocculation and sedimentation: SVE showed no visible flocculation, whereas SLP and SCA exhibited slight flocculation; additionally, moderate sedimentation was observed only in SCA. Although these findings indicate some differences in mechanical behavior among formulations, no complete compaction, large aggregates, or marked phase separation were observed, suggesting that the powder systems retained their overall macroscopic integrity under the evaluated centrifugal stress (29). Overall, the results support the preliminary mechanical stability of the developed powder prototypes under the evaluated centrifugation conditions. 4.2.4 Long-term stability test The long-term stability test performed at room temperature for 9 months showed preservation of several macroscopic and physical characteristics of the SLP, SCA, and SVE formulations (Tables 11– 13). Visual attributes such as color and general uniformity remained stable throughout the study, indicating preservation of the overall macroscopic appearance of the formulations. Slight decreases in texture, powder flow, and absence-of-lumps scores were observed after approximately 3 months, which may reflect progressive changes in interparticle interactions and powder cohesion during storage (29). In EO-containing formulations, aroma intensity decreased over time, which is compatible with partial loss of volatile constituents, although the characteristic aroma remained detectable. Such changes are plausible because EO components may undergo volatilization, oxidation, or degradation during storage depending on environmental and formulation conditions (38). Overall, the observed variations were gradual and were mainly associated with powder-handling properties and aroma rather than visible loss of formulation uniformity. The consistency between the long-term and accelerated thermal observations further supports the interpretation that the prototypes retained preliminary physical stability under the evaluated conditions (29, 31). Together with the pH and centrifugation assessments, these findings provide complementary evidence of the physical behavior of the formulations under the evaluated conditions, although moderate changes in texture, flow behavior, and aroma were observed over time. An additional limitation of the present study is that the chemical stability of the EO constituents was not quantitatively assessed during storage. Although the formulations maintained their main physical and organoleptic characteristics under the evaluated conditions, these observations cannot establish preservation of the chemical composition of the incorporated EOs. Essential-oil constituents, particularly volatile compounds, may be affected by volatilization, oxidation, or chemical degradation during storage (38). Future studies should therefore include quantitative GC–MS profiling of representative volatile constituents at baseline and after defined storage periods to characterize compositional changes and more accurately establish formulation shelf life (31). 4.2.5 Complementary physicochemical characterization The complementary physicochemical analyses provided additional objective information on the technological properties of the selected dry shampoo prototypes. SEM examination showed a heterogeneous particulate morphology in SCA, SLP, and SVE, with granular structures and particle aggregates observed across the formulations. These findings are consistent with the heterogeneous nature of multicomponent powder systems, in which differences in particle morphology, surface characteristics, and interparticle interactions may contribute to cohesion and powder-flow behavior (26, 28, 42). However, SEM observations were qualitative and should not be interpreted as a quantitative assessment of particle-size distribution or as direct evidence of the distribution of individual formulation components. The Hausner ratio and Carr index values obtained for all three formulations were consistent with cohesive powder behavior and limited intrinsic flowability. SCA and SLP showed similar values, whereas SVE presented the highest HR and CI values. These objective measurements complement the qualitative observations obtained during formulation development and suggest that the final formulations remained cohesive powder systems and may require further optimization for reproducible handling and potential scale-up (26, 27, 29). Moisture contents ranged from 10.69 to 14.02%; however, because moisture content was determined once for each formulation, these values should be interpreted descriptively rather than as evidence of differences among formulations. Moisture is also relevant to powder
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science17frontiersin.org performance because it can modify interparticle interactions, agglomeration, and flow behavior during storage. FTIR analysis showed broadly similar spectral profiles among SCA, SLP, and SVE, with the spectra of the finished formulations predominantly characterized by features shared with the common powder matrix. The absence of major new absorption bands after EO incorporation suggests that no marked changes in the overall infrared profile of the formulations were detected. Nevertheless, this observation does not exclude more subtle physicochemical interactions between the EOs and excipients, and FTIR alone cannot establish chemical compatibility or stability (43). DSC thermograms similarly showed comparable overall thermal profiles among SCA, SLP, and SVE, with no marked additional thermal event attributable exclusively to EO incorporation. These findings suggest that incorporation of CA-EO or LP-EO did not produce major detectable changes in the thermal behavior of the powder matrix within the evaluated temperature range. However, the absence of a distinct EO-related thermal event should not be interpreted as evidence that the volatile EO constituents remained chemically unchanged. FTIR and DSC can provide complementary information on spectral characteristics and thermal behavior within formulated systems (43), but they do not replace direct quantitative assessment of chemical stability. Accordingly, longitudinal chemical profiling, such as GC–MS, would still be required to establish the compositional stability of the EO constituents during storage (31, 38). Particle-size distribution and angle of repose were not directly determined in the present study. In particular, the SEM analysis provided qualitative information on particle morphology and aggregation but should not be interpreted as a quantitative particle-size distribution measurement. Future formulation-development and scale-up studies should incorporate these additional powder-characterization parameters to provide a more comprehensive assessment of flow behavior and manufacturing reproducibility. 4.3 In vitro antibacterial activity of the final formulations Direct evaluation of the finished dry shampoo formulations showed assay-dependent antibacterial behavior. In the agar diffusion assay, neither SCA nor SLP produced measurable inhibition zones against S. pseudintermedius ATCC 49444 or the three clinical isolates evaluated, whereas the 2% chlorhexidine control produced inhibition zones of approximately 20–24 mm. This contrasts with the previously reported activity of the isolated CA-EO and LP-EO against S. pseudintermedius (23), indicating that antibacterial activity demonstrated for an isolated EO cannot necessarily be extrapolated to the finished formulation. Incorporation into a solid matrix may influence the availability, release, and diffusion of lipophilic EO constituents, while agar diffusion itself depends strongly on the ability of antimicrobial compounds to migrate through the agar medium (10, 38, 44). Therefore, the absence of measurable inhibition zones should be interpreted in the context of the formulation and assay rather than as definitive evidence of complete absence of antibacterial activity. In contrast, the serial dilution experiments showed differences in viable bacterial recovery as formulation concentration decreased. SCA and SLP produced absence or marked reduction of detectable bacterial growth at higher tested concentrations, followed by increasing CFU/mL at greater dilutions, supporting a concentration-dependent response under liquid-phase conditions. Dilution-based susceptibility assays provide greater direct contact between microorganisms and the tested antimicrobial system and therefore complement diffusionbased approaches (45). Importantly, SVE also reduced bacterial recovery at several corresponding formulation dilution levels. This finding indicates that the observed effect cannot be attributed exclusively to CA-EO or LP-EO and suggests that the vehicle matrix or the physicochemical conditions generated by its components contributed to bacterial growth inhibition. Accordingly, interpretation of the MIC and CFU results should consider the complete formulation rather than the EO concentration alone. Taken together, the discrepancy between agar diffusion and serial dilution results emphasizes the influence of the experimental system on the apparent antimicrobial performance of complex topical formulations. Previous studies have demonstrated antibacterial activity of different EOs against staphylococci, including methicillin-susceptible and methicillin-resistant S. pseudintermedius isolates (46, 47), but the present results suggest that incorporation into a dry powder matrix may modify the antimicrobial behavior observed with the isolated oils. Thus, the current findings provide preliminary evidence of antibacterial activity under specific in vitro conditions, rather than definitive confirmation of antimicrobial efficacy of the finished shampoos. Further studies should evaluate a larger collection of clinical isolates and incorporate complementary approaches such as formulationrelease studies, time-kill assays, and antibiofilm models to distinguish more clearly the contributions of the EO and vehicle matrix to the observed antibacterial response. 4.4 Safety test in murine model Following macroscopic physicochemical characterization, the three final formulations (SLP, SCA, and SVE) were subjected to in vivo testing to assess their preliminary dermal safety. A murine model was used for this purpose. 4.4.1 Pre-clinical safety assessment Photographic monitoring of representative animals during the five-day experimental period (Figure 4) allowed evaluation of the dermal response at the application site. Throughout the study, the skin remained intact with no visible signs of irritation, including erythema, edema, ulceration, exudate, severe desquamation, open lesions, crusts, bleeding, or necrosis. Minor variations in skin coloration observed among individuals were not consistent or progressive and were therefore attributed to normal physiological differences or handling during the experimental procedure (18, 20). General welfare parameters— including appearance, food and water intake, body temperature, respiratory and heart rate, and spontaneous behavior—remained within normal ranges throughout the observation period. Body weight fluctuations were minimal, generally remaining below 5%, and showed no consistent trends among animals or treatment groups, which is compatible with normal physiological variation in young animals adapting to experimental conditions (17, 19). 4.4.2 Histological analysis Histological examination of skin samples collected at the end of the trial revealed preserved tissue architecture across the SLP, SCA, and SVE groups (Figure 5), with a continuous epidermis and well-defined dermal layers. No signs of ulceration, erosion,
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science18frontiersin.org necrosis, or hyperplasia were observed. The dermis displayed normal collagen fiber organization without evidence of inflammatory infiltration or edema, with no histological evidence of treatment-associated cellular damage or inflammatory responses under the evaluated conditions. Comparable histological features were observed in all groups, including the vehicle formulation, suggesting that the shampoo base itself did not produce detectable tissue alterations under the evaluated conditions. In particular, the EO-containing formulations (SLP and SCA) exhibited tissue responses similar to the vehicle, suggesting that EO incorporation did not introduce histological changes under the tested conditions. Together with the clinical observations, these findings provide preliminary evidence that the developed prototypes were well tolerated under the evaluated topical exposure conditions and support further dermal safety evaluation for potential veterinary application (18, 20, 48). The preclinical dermal safety evaluation in BALB/c mice provided additional evidence supporting the tolerability of the formulations. Repeated topical applications did not produce overt adverse clinical signs, behavioral changes, erythema, edema, or histological evidence of inflammation or tissue damage under the evaluated conditions. This finding is relevant because EOs may exert biological effects in mammalian cells depending on their chemical composition and concentration; therefore, safety assessment is an essential step in the development of EO-based topical products (49). However, the results obtained in mice should be considered as preliminary preclinical evidence and do not replace safety evaluation in canine patients under clinical conditions. An additional limitation of the present study is that dermal permeation and systemic exposure to EO constituents were not directly assessed; consequently, the extent of transdermal absorption following topical application cannot be established from the current findings. Although local dermal tolerability and general safety parameters were monitored, these assessments cannot substitute for dedicated permeation or pharmacokinetic studies. Future investigations should therefore include in vitro/ex vivo permeation experiments using excised canine skin and diffusion-cell systems following the principles of OECD Test Guideline 428, allowing quantitative determination of representative EO constituents retained within the skin and those reaching the receptor compartment, thereby providing information on dermal retention and transdermal absorption. Assessment of skin barrier integrity before and after exposure should also be incorporated to determine whether repeated application affects cutaneous barrier function, while complementary quantification of representative EO constituents in plasma following repeated topical administration would help characterize systemic exposure and bioavailability. Together, these approaches will be important to determine whether exposure remains predominantly local and to more comprehensively characterize the safety profile of the formulations under repeated topical use. 4.5 Microbiological evaluation in dogs The microbiological assessment in dogs offered an initial in vivo approximation of the antimicrobial behavior of the dry shampoo formulations under clinical conditions. To characterize the response more comprehensively, three outcomes were considered: bacterial presence or absence, bacterial load categories, and classification of growth as significant or non-significant. Before treatment, nearly all dogs yielded positive cultures for Staphylococcus spp., supporting the infectious nature of the lesions and agreeing with the recognized predominance of S. pseudintermedius in canine superficial pyoderma (2, 50). By Day 21, absence of detectable cocci in culture was recorded in the SCA and Inveclor ® groups, whereas all evaluable dogs in the SLP and SVE groups remained culture positive. The highest clearance frequency was observed in the Inveclor ® group, which is expected considering the established role of chlorhexidine-based topical products in the treatment of canine superficial pyoderma (2, 51). Among the essential-oil-loaded dry shampoos, SCA was the only EO-containing formulation in which culture negativity was observed, suggesting a more favorable culture-based response for the C. alba EO formulation. Bacterial load categorization allowed the response to be interpreted with greater resolution than culture positivity alone. On Day 21, the SCA group had the largest proportion of dogs classified within the non-significant bacterial load category, followed by Inveclor ® , SVE, and SLP. In the case of the SLP group, one dog showed nonsignificant bacterial growth at Day 21, while significant bacterial growth was observed in the remaining cases. In addition, SLP showed the highest proportion of dogs in the uncountable category, which suggests that the antibacterial activity previously observed in vitro for L. philippiana EO was not clearly reproduced in the culture-based in vivo evaluation. The categorization of bacterial growth as significant or non-significant supported this trend, with SCA showing the highest proportion of dogs with non-significant bacterial growth at Day 21. However, comparisons among treatment groups did not reveal statistically significant differences. Therefore, these results should be interpreted as exploratory trends rather than definitive evidence of treatment efficacy. Nevertheless, bacterial load and growth categorization may represent useful endpoints for future studies, as they allow the microbiological response to be evaluated beyond a binary presence/ absence outcome. This distinction is clinically relevant because the management of canine superficial pyoderma does not depend exclusively on complete bacterial eradication. Reduction of bacterial burden, limitation of lesion progression, improvement of the skin microenvironment, and support of barrier recovery are also important therapeutic goals. Current recommendations emphasize diagnosis supported by cytology, bacterial culture when indicated, topical therapy for surface and superficial infections, and responsible antimicrobial use to reduce unnecessary systemic antibiotic exposure (2, 6). Within this framework, a topical product that helps reduce bacterial burden may be valuable even when full microbiological clearance is not achieved in every patient. The comparatively better microbiological profile observed for SCA may be associated with the chemical composition of C. alba EO. This oil contains monoterpenes such as sabinene, eucalyptol, terpinen-4-ol, α-pinene, and β-pinene, which have been related to antibacterial effects involving membrane disruption, increased cell permeability, leakage of intracellular components, and interference with bacterial adhesion or persistence (10, 14, 52). Comparable mechanisms have been reported for other essential oils tested against staphylococci, including S. pseudintermedius isolates associated with canine pyoderma (46, 47).
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science19frontiersin.org The differences observed between the assay-dependent in vitro performance of the finished formulations and the microbiological responses obtained in vivo further illustrate the difficulty of extrapolating laboratory antimicrobial results to clinical settings. In dogs, bacterial dynamics are shaped by lesion characteristics, skin barrier condition, local inflammation, host immunity, grooming behavior, environmental exposure, owner adherence, and retention of the formulation on the skin. These variables are especially relevant in superficial pyoderma, which commonly occurs secondary to barrier dysfunction, allergic disease, or recurrent dermatological disorders (50, 53). For this reason, the present dog trial should be regarded as exploratory and hypothesis-generating rather than definitive. These results should also be interpreted in the broader context of antimicrobial resistance. S. pseudintermedius is a key canine pathogen, and methicillin-resistant or multidrug-resistant strains remain a major concern in veterinary medicine (1, 54, 55). In the present study, none of the recovered S. pseudintermedius isolates showed phenotypic resistance to oxacillin. However, this finding should be interpreted cautiously, as methicillin resistance was assessed phenotypically and was not confirmed by molecular detection of resistance determinants such as mecA. Thus, the results indicate the absence of phenotypically detected MRSP rather than the definitive exclusion of methicillin resistance. Therefore, the development of well-tolerated topical alternatives may contribute to One Healthoriented strategies aimed at reducing reliance on systemic antimicrobials, particularly considering the potential zoonotic circulation of S. pseudintermedius (3, 56). Several limitations should be acknowledged. Although the antimicrobial activity of the final dry shampoo formulations was directly evaluated using agar diffusion, serial dilution, MIC assessment, and quantitative viable bacterial counts, these analyses were preliminary and included only S. pseudintermedius ATC C 49444 and three clinical isolates. Therefore, the observed antibacterial responses cannot yet be considered representative of the broader bacterial population associated with canine superficial pyoderma (46, 47). Moreover, because SVE also reduced bacterial recovery at several corresponding formulation dilution levels, the contribution of the vehicle matrix cannot be excluded and the observed in vitro activity cannot be attributed exclusively to the incorporated EOs. The clinical study was designed to detect large microbiological effects but was not powered to detect small or moderate treatment effects or to establish definitive clinical efficacy, equivalence, or non-inferiority. In addition, microbiological outcomes were not integrated with a standardized quantitative lesion-severity score, and the individual contribution or potential synergistic interactions of the EO constituents were not experimentally determined. Additional limitations are related to the clinical heterogeneity of the enrolled canine population. Dogs were recruited under realworld conditions from multiple veterinary centers, and a substantial proportion were mixed-breed animals. Although eligibility criteria required a minimum 15-day treatment-free period for systemic or topical antibiotics, corticosteroids, and natural products with potential antimicrobial or anti-inflammatory activity, complete standardized information regarding breed or breed type and dermatological treatments received before this exclusion period was not available for all animals. Consequently, these variables could not be incorporated into treatment-group stratification or statistical adjustment and may have contributed to variability in microbiological response. Furthermore, clinical follow-up was limited to the 21-day treatment period; therefore, persistence of the microbiological response, recurrence of superficial pyoderma, and longer-term tolerability after treatment discontinuation could not be determined. Future studies should therefore include a larger and more diverse collection of clinical isolates, complementary time-kill, antibiofilm, formulation-release, and skin-retention studies, and larger randomized controlled clinical trials using standardized clinical outcome measures (2, 44, 50). Overall, although the antibacterial performance of the finished formulations was assay-dependent and the between-group microbiological differences observed in vivo were not statistically significant, the incorporation of native plant EOs into a dry shampoo platform with preliminary physical stability and favorable preclinical tolerability represents a relevant formulation-development step. Among the EO-containing formulations, SCA showed the most favorable exploratory microbiological profile, with the highest proportion of dogs reaching a non-significant bacterial burden and the only cases of culture negativity observed among the EO-loaded shampoo groups. These findings support further investigation of C. alba EO-containing dry shampoos as potential topical candidates for canine superficial pyoderma, while emphasizing that larger controlled studies are required before conclusions regarding clinical efficacy can be established (2, 23). 5 Conclusion This study reports the development and characterization of starch-based dry shampoo prototypes incorporating EOs from L. philippiana and C. alba, together with the assessment of their in vitro antibacterial performance, preclinical dermal tolerability, and exploratory microbiological response in dogs with naturally occurring superficial pyoderma associated with S. pseudintermedius. Overall, the different experimental stages addressed the central hypothesis that these EOs could be incorporated into dry shampoo formulations with preliminary physical stability, acceptable local dermal tolerability under the evaluated conditions, and measurable microbiological responses. The use of native Chilean plants as sources of bioactive compounds represents a potential strategy for the development of complementary topical approaches in veterinary dermatology. From a formulation perspective, incorporation of magnesium stearate and selection of an appropriate mixing protocol improved powder homogeneity, reduced agglomeration, and enhanced handling and flow-related characteristics. Complementary physicochemical characterization further showed that the selected prototypes remained cohesive powder systems and exhibited preliminary physical stability under the evaluated conditions. However, the alkaline pH, absence of batch-to-batch reproducibility assessment, and lack of longitudinal chemical profiling remain relevant formulation limitations. Direct in vitro evaluation of the finished formulations revealed assay-dependent antibacterial behavior. No measurable inhibition
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science20frontiersin.org zones were observed by agar diffusion, whereas serial dilution and viable-count experiments showed concentration-dependent reductions in bacterial recovery. Because inhibitory effects were also observed with the vehicle formulation, the antibacterial response cannot be attributed exclusively to the incorporated EOs. These findings highlight the importance of evaluating the complete formulation rather than extrapolating antimicrobial activity from the isolated EOs alone. The preclinical safety assessment showed that repeated topical application of the EO-containing formulations and vehicle in BALB/c mice did not produce visible erythema, edema, relevant behavioral or body-weight alterations, or histological evidence of treatment-associated inflammation or tissue damage under the evaluated conditions. These findings provide preliminary evidence of local dermal tolerability but do not replace safety assessment in the target species or dedicated studies of dermal permeation and systemic exposure. In dogs with superficial pyoderma, numerical differences were observed among treatment groups in bacterial detection, bacterialload categories, and significant versus non-significant growth. These differences were not statistically significant and should not be interpreted as evidence of clinical efficacy. Nevertheless, among the EO-containing formulations, SCA showed the most favorable exploratory microbiological profile, including the highest proportion of dogs reaching a non-significant bacterial burden and the only cases of culture negativity observed among the EO-loaded shampoo groups. The leave-on dry-powder format may be particularly advantageous for localized lesions, intertriginous areas, or dogs in which repeated bathing is difficult or impractical, potentially simplifying repeated topical administration and reducing handling requirements. However, these practical advantages were not formally evaluated in the present study and should be confirmed in future usability and owner-adherence studies. Taken together, these findings support the feasibility of incorporating L. philippiana and C. alba EOs into starch-based dry shampoo prototypes and provide preliminary evidence of physical stability, preclinical dermal tolerability, and assay-dependent antibacterial and exploratory microbiological performance. The leaveon powder format may offer practical advantages, including localized application and administration without rinsing, although these potential benefits were not formally evaluated. Further work should include pH optimization, independently prepared formulation batches, quantitative longitudinal chemical stability profiling, formulation-release, time-kill and antibiofilm studies, dermal permeation and skin-retention assessment, larger randomized controlled clinical trials incorporating validated clinical scoring systems, and extended safety evaluation before conclusions regarding clinical efficacy or long-term veterinary use can be established. 6 Patents The national registration application for the antibacterial dry shampoo has been filed with INAPI Chile and is currently under review for acceptance. Data availability statement The original contributions presented in this study are included in the article/Supplementary materials. Further inquiries can be directed to the corresponding author. Ethics statement The animal studies were approved by the Ethics Committee of the Odontology Faculty of the University of Chile (protocol 24814-ODO- UCH-e3; 4 June 2025) and the Ethics Committee of Universidad Mayor (protocol ID14/2024; 28 June 2024). The studies were conducted in accordance with local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study. Author contributions MV: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. MJ: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. NR: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. PR: Investigation, Methodology, Project administration, Resources, Writing – original draft. FB: Conceptualization, Methodology, Writing – original draft. GV-B: Conceptualization, Validation, Visualization, Writing – review & editing. OC: Conceptualization, Validation, Writing – original draft. AD-P: Investigation, Methodology, Writing – original draft. MA: Formal analysis, Writing – review & editing. GM: Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. IM: Visualization, Writing – review & editing. FA: Investigation, Methodology, Validation, Writing – original draft. BR: Investigation, Methodology, Validation, Writing – original draft. MB: Investigation, Methodology, Validation, Writing – original draft. DS: Conceptualization, Project administration, Validation, Visualization, Writing – review & editing. JB: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Funding The author(s) declared that financial support was received for this work and/or its publication. This work was supported by IDEA ID24I10250; INGE 230004; CIA250002–CEDENNA. Acknowledgments The authors would like to thank the veterinary teams of the Oftaderm Veterinary Clinic, the Veterinary Teaching Hospital of Universidad Mayor, and the municipal veterinary clinics of La Granja
Varas et al. 10.3389/fvets.2026.1918882 Frontiers in Veterinary Science21frontiersin.org and Conchalí (Santiago, Chile) for their valuable collaboration in the recruitment and clinical management of the canine patients included in the preliminary microbiological evaluation. Their support and commitment were essential for the development of the clinical component of this study. Conflict of interest FA and BR were employed by Oftaderm Veterinary Clinic. The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Generative AI statement The author(s) declared that Generative AI was used in the creation of this manuscript. The authors acknowledge the use of ChatGPT, based on GPT-5.5 Thinking, developed by OpenAI, for language editing, grammar revision, and formatting support during manuscript preparation. All AI-assisted outputs were revised and verified by the authors. The authors take full responsibility for the content, accuracy, originality and integrity of the final manuscript. Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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