Formulation of menthol for sedation and anesthesia of fish

Abstract

The present invention relates to new liquid compositions, formulated to keep menthol as an anesthetic agent stable in the liquid composition, and to the use of this composition. The composition is for sedation or anesthesia of fish in different handling procedures including in fish breeding and harvesting facilities. The invention relates to liquid compositions for anesthesia of fish, and particularly to compositions comprising a menthol as an anesthetic agent, and wherein the menthol is formulated in a liquid vehicle comprising propylene glycol. Furthermore, the invention relates to a method for anesthesia of fish, using the liquid composition comprising a menthol.

Description

FORMULATION OF MENTHOL FOR SEDATION AND ANESTHESIA OF FISHTechnical Field

[0001] The present invention relates to compositions for anesthesia of fish, and particularly to liquid compositions comprising a menthol as an anesthetic agent. Furthermore, the invention relates to a method for anesthesia of fish, using the liquid composition comprising a menthol formulated in a propylene glycol. The formulated liquid composition is directly miscible in water.Background Art

[0002] Sedation or anesthesia of fish is needed in connection with various operating operations, such as in handling procedures like crowding, treatments and transport, sealice counting, vaccination and harvesting of farmed fish.

[0003] The use of chemicals is one alternative for anesthesia and a range of anesthetic agents are currently in use in laboratory, veterinary, and aquaculture contexts. Common anesthetic drugs used for fish are MS-222 (Tricaine), benzocaine, clove oil (eugenol), isoeugenol, metomidate, 2-phenoxyethanol, and quinaldine.

[0004] Menthol is known for its anesthetic properties and has been tested as a sedative and / or anesthetic on several tropical species of fish, largely by Brazilian researchers. In addition, one South African study has investigated the effect on rainbow trout; C Teta & H Kaiser (2019): Menthol as an alternative anesthetic and sedative for rainbow trout, Oncorhynchus mykiss, Africa Journal of Aquatic Science, using stock solutions of menthol dissolved in ethanol or acetone. Formulations with ethanol are flammable and / or explosive and entail restrictions on transport and handling. Furthermore, the stability of menthol in an ethanol formulation is unknown or not documented.

[0005] There is hence a need for more user-friendly and improved methods for anesthesia of fish.Summary of inventionThe invention provides an anesthetic liquid composition comprising menthol and user-friendly and improved methods using the menthol composition for anesthesia of fish.In one aspect, the invention provides an anesthetic liquid composition comprising: i) 20-70% by weight of an anesthetic agent selected from the group of menthols; ii) 30-80% by weight of a liquid vehicle comprising at least one excipient, wherein the at least one excipient comprises a propylene glycol.More particularly, the invention provides an anesthetic liquid composition comprising: 20-70% by weight of an anesthetic agent selected from the group of menthols; 20-60% by weight of an excipient being a propylene glycol;0.1-20% by weight of a further excipient selected from non-ionic surfactants from the group of polysorbates and ethoxylated emulsifying agents.In one embodiment, the liquid composition comprises at least one further excipient selected from non-ionic surfactants. The at least one further excipient is selected from non-ionic surfactants from the group of polysorbates and ethoxylated emulsifying agents, preferably macrogolglycerol hydroxystearate, such as in a weight% of 0.1-20% of the composition.

[0006] In another aspect, the invention provides a composition for use in anesthetic treatment of fish, the use comprises a step of adding a liquid composition according to the first aspect to a water container comprising the fish.Figures

[0007] Figure 1 shows the average sedative effect of a composition of the invention comprising 500 mg menthol / g (“Calmenthol”) in two different low dosages (3.10 mg / l and 5.15 mg / l) compared with Aqui-S (2.48 mg isoeugenol / l) on Atlantic salmon (n = 10) over time (minutes) while exposed to an anesthetic bath for 30 minutes at 5°C.Figure 2 shows the average sedative effect of Calmenthol in two different low dosages (3.10 mg / l and 5.15 mg / l) compared with Aqui-S (2.48 mg isoeugenol / l) on Atlantic salmon (n = 10) over time (minutes) while exposed to an anesthetic bath for 30 minutes at 15°C.Figure 3 shows the average anesthetic effect at 5°C of Calmenthol in two different high dosages (82.7 mg / l and 165.3 mg / l) compared with effect of Tricaine mesylate (TMS) (100 mg / l) over time (seconds). Atlantic salmon (n=10) were exposed until surgical anesthesia was reached, then transferred to fresh water for recovery.Figure 4 shows the average anesthetic effect at 15°C of Calmenthol in two different high dosages (82.7 mg / l and 165.3 mg / l) compared with effect of Tricaine mesylate (TMS) (100 mg / l) over time (seconds). Atlantic salmon (n=10) were exposed until surgical anesthesia was reached, then transferred to fresh water for recovery.Detailed description of the invention

[0008] The invention provides a composition for anesthesia of fish. Anesthesia is a biological condition with the partial or complete loss of sensation, responsiveness, and loss of voluntary neuromotor control induced by chemical or non-chemical means. Fish feel less pain and become calm during anesthesia thus, anesthesia is a useful, and required, means for fish handling, for transportation, for surgery, and before slaughter. The term “anesthesia” used herein covers different degrees of loss of sensation, responsiveness, and loss of voluntary neuromotor control, i.e., from light sedation to complete anesthesia.

[0009] The provided composition is liquid and comprises an anesthetic agent as active ingredient, wherein this is preferably a menthol. The provided composition is to be added directly to water containing fish to be sedated or anesthetized. The composition is hence for use in the process of sedation of fish during handling procedures like crowding, treatments and transport, as well as full anesthesia of fish during procedures like sealice counting, vaccination and harvesting of farmed fish. As a result, the stress induced on the fish is reduced, providing improved fish welfare and better product quality. Instead of today’s methods for anesthesia of fish, e.g. prior to slaughter, which is largely electrical or mechanical stunning, using the composition of the invention the fish is anesthetized in a bath containing solubilized anesthetic agent.Provided herein is a liquid formulated composition comprising an anesthetic agent suitable for anesthesia, i.e. of either sedation or anesthesia of fish, and at least one excipient comprising a propylene glycol. In one embodiment, the anesthetic agent istypically a menthol. In an alternative embodiment, the anesthetic agent is selected from the group of menthols, phenol, eugenol and isoeugenol. The components of the liquid composition, i.e. , the anesthetic agent and the at least one excipient are recognized as safe to use with respect to food safety, as the primary use of the composition is for sedation or anesthesia of fish in different handling procedures in fish breeding and harvesting facilities, i.e. of fish meant for human consumption. The liquid composition is efficacious to anesthetize fish and has beneficial health effects including suppression of physiological and oxidative stress. The liquid composition can be added directly to the water without need for making a stem solution, unlike some of the other anesthetic formulations on the market.

[0010] According to one aspect of the invention, there is provided a liquid composition comprising menthol in a concentration of at least 20 weight% of the composition, further comprising at least one excipient comprising a propylene glycol.

[0011] Accordingly, the invention provides an anesthetic liquid formulated composition comprising: i) 20-70% by weight of an anesthetic agent selected from the group of menthols; ii) 30-80% by weight of a liquid vehicle comprising at least one excipient, wherein the excipient comprises a propylene glycol.In one embodiment, the composition comprises a further excipient, such as in a weight% of 0.1-20, in addition to the propylene glycol. The further excipient is preferably a non-ionic surfactant. In one embodiment, the at least one further excipient is macrogolglycerol hydroxystearate. The at least one excipient has at least the function of solubilizing the anesthetic agent, increasing the water solubility of the menthol.

[0012] The composition is primarily provided for use in sedation or anesthesia of farmed fish. Hence, an anesthetic liquid composition is provided formulated to keep the anesthetic agent stable as a liquid composition. The composition is accordingly a fish anesthetic composition.

[0013] In one embodiment, the anesthetic agent of the composition is a menthol. Menthol is a monoterpenoid, which can be made synthetically or be obtained from oils of corn mint, peppermint, or other mints. The chemical name of menthol is 2-isopropyl-5-methylcyclohexanol, 5-methyl-2-(propan-2-yl)-cyclohexan-1-ol and is also called menthyl alcohol. The formula of menthol is C10H20O and the molecular weight is 156.27 g / mol. Menthol is a waxy, clear, or white crystalline substance, which is solid at room temperature and melts slightly above room temperature. The main form of menthol occurring in nature is (-)-menthol, which is assigned the (1 R,2S,5R) configuration, however 8 stereoisomers exist. There are four diastereomeric menthols based on the relative orientations of the methyl, isopropyl, and hydroxyl groups, and these are known as menthol, isomenthol, neomenthol, and neoisomenthol, each of which exists in two enantiomeric forms. The menthol of the composition may have any of the stereoisomeric forms. In a preferred embodiment, the menthol is L-menthol, also called Levomentol, or (1 R, 2S,5R)-2-isopropyl-5- methyl, with CAS Registry No. 2216-51-5, which is a pure stereoisomer of natural menthol.

[0014] In one embodiment, the menthol of the composition is a registered monographed substance, ensuring high quality, correct identity, high purity and performance. Preferably the menthol of the composition is the registered L-menthol (Levomenthol) with monograph No. 619, Certificate of Suitability of European Pharmacopoeia monographs (CEP), in the EDQM Database. In one embodiment, the enantiomeric purity is >99.7%, the chromatographic purity is >99.7 area% and / or the degree of impurities are < 0.3 area%.

[0015] The liquid composition of the invention is formulated such that menthol, which is a crystalline solid substance at room temperature, is completely solved in a liquid vehicle comprising the at least one excipient, providing the composition as a clear, liquid solution. It is an advantage of the invention that menthol as an anesthetic agent is provided in the form of a liquid composition which can be added directly to the water wherein the fish is. The liquid anesthetic composition is miscible with water, and when added to the water where the fish is, the liquid composition is completely miscible with the water. The liquid composition of the invention enables a user- friendly delivery of the active substance menthol as a stable formulation, as this can be added directly to water and which is easily mixed directly in water. There is no need for preparation of a stem solution. The liquid anesthetic composition prevents menthol precipitation, and thereby reduces loss of menthol to the surroundings. Nomenthol crystal formation is seen when adding the composition to representative water vessels.

[0016] The at least one excipient of the liquid vehicle comprises a solvent that is completely soluble in water and having the ability to solve menthol. Hence, the excipient(s) are, or is part of, a liquid vehicle which function to solubilize the anesthetic agent menthol, increasing the water solubility of the menthol, and to provide a maximum menthol load with an acceptable viscosity.

[0017] In one embodiment, the at least one excipient comprises a propylene glycol. An ethylene glycol could alternatively be used, although this is not such a good solvent. Propylene glycol, which is a propanediol, has two hydroxyl groups and have intermediate properties between alcohols and glycerin and the solubility characteristics of glycols tend to be between those of simple alcohols and glycerin. In one embodiment, the at least one excipient is a solvent, alternatively seen as a carrier, selected from the group of propylene glycol, di-propylene glycol and tripropylene glycol. As the simplest propylene glycol, this may be selected from 1 ,2- propanediol (alpha-propylene glycol) and 1 ,3-propanediol (beta-propylene glycol). In a preferred embodiment, the excipient comprises propylene glycol, and is preferably 1 ,2-propanediol. The propylene glycols are found useful as they have good solvent properties for menthol, as they are completely soluble in water, as they have a high boiling point, and a low freezing point. In one embodiment, the at least one excipient is the propylene glycol named Kollisolv PG®.

[0018] To solubilize the menthol, and formulate this into a stable, liquid composition, the composition may comprise a further excipient useful to solubilize water-insoluble active substances such as menthol. In one embodiment, the liquid vehicle of the composition comprises an excipient selected from non-ionic surfactants, in addition to the propylene glycol. In one embodiment, the at least one further excipient is selected from non-ionic surfactants, particularly from the group of polysorbates and ethoxylated emulsifying agents. The further excipient(s) accordingly functions as a solubilizer, emulsifier and / or a surfactant. Selected excipients of the composition are further recognized as safe to use with respect to food safety. Particularly, as an intended use of the anesthetic composition is for anesthesia of food producing fish, components are selected that comply with regulations provided for veterinarymedicines intended for use in food producing animals (Regulation (EC) No 470 / 2009). The formulated composition has been arrived at by considering an intended use in food producing fish, and components have been selected also to comply with governmental regulations directed to the use of medicinal products on food producing fish, including food safety and environmental issues of components, and on governmental regulations regarding stability and documentation of medicinal products.

[0019] The optional further excipient(s) may be based on castor oil, or another vegetable oil, or is selected from ethoxylated surfactants, such as for instance ethoxylated sorbitan esterified with fatty acids (e.g. polysorbates). In one embodiment, the further excipient is an ethoxylated emulsifying agent of the type wherein ethylene oxide is reacted with hydroxy stearic acid, such as macrogol 15 hydroxystearate. Advantageously, the further excipient is selected from the group of polysorbates, such as Polysorbate 20, 40, 60 or 80, or mixtures thereof, and from excipients based on castor oil, such as macrogolglycerol hydroxystearate. The latter is an agent obtained from ethylene oxide reacted with hydrogenated castor oil and is hence an ethoxylated castor oil. The ethoxylation of castor oil enhances the solubility and emulsifying properties of the castor oil. The IIIPAC name is 2-[1 , 3-bis[2-(11 - hydroxyheptadecoxycarbonyloxy)ethoxy]propan-2-yloxy]ethyl 11 -hydroxyheptadecyl carbonate, CAS number 61788-85-0. In one embodiment, the at least one further excipient is macrogolglycerol hydroxystearate, such as the one named Kolliphor® RH40. In one embodiment, if a non-ionic surfactant comprised in the composition is a polysorbate this may be either of Kolliphor® PS 20, Kolliphor® PS 60 or Kolliphor® PS 80. In yet another embodiment, if the further excipient comprised in the composition is a solubillizer based on ethylene oxide reacted with hydroxy stearic acid this may be Kolliphor® HS 15.

[0020] The liquid composition comprises the anesthetic agent in suitable, effective concentrations. Suitably, liquid composition comprises the anesthetic agent in concentrations of at least 20% by weight, which is a concentration suitable for use, such as for adding to a water tank of fish to be anesthetized. As menthol is a crystalline substance, solid at room temperature, this of course has a solubility concentration limit in the liquid vehicle. The composition typically comprises theanesthetic agent in a weight% below the solubility limit of menthol (solute) in the liquid vehicle (at least one excipient), ensuring a liquid clear solution. The composition typically comprises the anesthetic agent in about 20-70 weight%, preferably in about 30-60 weight%, and most preferably in about 40-55 weight%.

[0021] The liquid composition comprises the aqueous vehicle in about 30 - 80 weight%, such as in about 40 - 70 weight%. The aqueous vehicle comprises at least one excipient comprising a propylene glycol. The concentration of the liquid vehicle comprising at least one excipient is adequate to completely solve the anesthetic agent. In one embodiment, the composition comprises a propylene glycol in about 20-80 weight% of the composition, more preferably in about 20-60 weight% or 30-60 weight%, and most preferably in about 35-55 weight%.

[0022] In one embodiment, the liquid vehicle comprises an optional further excipient in the form of a non-ionic surfactant in a concentration of 0-20 weight% of the composition, such as in a concentration of 0-15 weight%, such as 0.1-20 weight%, preferably 5-15 weight%, such as around 10 weight% of the anesthetic liquid composition.

[0023] Advantageously, the liquid vehicle comprises an amount of polypropylene glycol and the optional additional excipient sufficient to completely solubilize the anesthetic agent in the composition. Furthermore, in addition to completely solubilize the anesthetic agent, the liquid vehicle also provides an adequate viscosity of the liquid composition. The optional additional excipient, such as a polysorbate or an ethoxylated castor oil, e.g. macrogolglycerol hydroxystearate, may have such function. As a result, the viscosity of the liquid composition is low, also even at temperatures around the freezing point, and is not more viscous than for example rapeseed oil. The viscosity of the liquid composition increases as the temperature is lowered, however this is still relatively low and acceptable even for temperature towards the freezing point. In one embodiment, the viscosity of the liquid composition is e.g. less than about 100 mm2 / s at 20 °C, less than about 150 mm2 / s at 15 °C, or less than about 290 mm2 / s at about 6 °C, measured as kinematic viscosity by the method according to Ph. Eur. 11thEd, 01 / 2019:20209 Capillary Viscometer Method. Reference is made to Example 1 , providing the viscosity of a formulation of the invention. Due to the low viscosity, the liquid composition can be added directly to acontainer such as a tank, net pen or well without leaving menthol film on surfaces or form any precipitates.

[0024] Accordingly, advantageous amounts of the components of the liquid composition are:- at least 20% by weight of the anesthetic agent;- a 20-60% by weight of a propylene glycol;- optionally a non-ionic surfactant, and if present this is in a weight% of 0.1-20.

[0025] More particularly, an anesthetic liquid composition is provided, the composition comprising20-70% by weight of an anesthetic agent selected from the group of menthols;20-60% by weight of an excipient being a propylene glycol;0.1-20% by weight of a further excipient selected from non-ionic surfactants, such as from the group of polysorbates and ethoxylated emulsifying agents.In a particularly preferred embodiment, the liquid composition comprises about 40- 55% by weight of menthol, and about 35-45% by weight of propylene glycol, preferably 1 ,2-propanediol. The composition preferably further comprises about 5- 15% by weight of at least one non-ionic surfactant. The at least one non-ionic surfactant is selected from polysorbates and macrogolglycerol hydroxystearate, and is preferably macrogolglycerol hydroxystearate.

[0026] Examples of tested liquid compositions according to the invention are provided below:1 ) Menthol 50.0 wt%, 1 ,2-propanediol 40 wt%, Kolliphor® PS 20 10 wt% (Polysorbate 20)2) Menthol 50.0 wt%, 1 ,2-propanediol 35 wt%, Kolliphor® HS 15 wt% (Macrogol- 15-hydroxystearate)3) Menthol 50.0 wt%, 1 ,2-propanediol 40 wt%, Kolliphor® RH 40 10 wt% (Macrogolglycerol hydroxystearat)All the above compositions have shown favourable results including no recrystallisation after 24 hours or one week at 4°C and 24 °C, and very low viscosity at 2, 4 and 24 °C.

[0027] An advantage of the anesthetic liquid composition is that this is in liquid form, and hence easily miscible with water. When used in a fish breeding or harvesting facility, this can simply be poured into the water of a container where the fish is. An advantage is that also if the water is cold, such as down to 4 °C, the composition will still blend easily with the water providing a very user-friendly method for anesthesia of fish. In addition to easiness of use, the overall cost is low, and it is effective. The composition is added to the container where the fish is kept inducing sedation and / or anesthesia. The fish container may be selected from the group of a well, a tarpaulin- enclosed cage, holding tank or similar. In one example the container is a well of a well boat.Appropriate dosages for use in anesthesia of fish are 2-320 mg menthol / liter water, such as 5-320 mg / liter, 80-320 mg / liter, such as 5- 160 mg / liter. The dosage would vary depending on the degree of sedation or anesthesia wanted. The degree of anesthesia may be classified into different stages, from stages 0-IV as provided below.Stage 0: AlertStage I: Light sedationStage II: ExcitationStage III: Plane 1 Light anesthesiaStage III: Plane 2 - surgical anesthesiaStage III: Plane 3 - deep narcosisStage IV: Imminent death - Moribund

[0028] If a lighter sedation is wanted, e.g. as a pre-anesthetic step, a lower dosage should be provided, e.g. in ranges of 2-20 mg menthol / liter water. If a direct anesthesia is wanted the composition is added to the water container at dosages typically ranging from 80 to 320 mg menthol / liter water. For sedation about 5 mg menthol / liter water is typically appropriate while for full anesthesia about 160 mg menthol / liter water is appropriate. Accordingly, with a composition comprising about 500 mg menthol per gram, a typical dosage is about 10 grams (e.g. 5-15 grams) ofthe liquid composition per 1000 liter of water for sedation and about 320 grams (e.g. 160-480 grams) per 1000 liter water for full anesthesia.

[0029] The composition of the invention may be provided in suitable containers with a manageable size for pouring of the composition. In one embodiment, the composition is provided in containers of 10-10 000 ml, such as in sizes of 100 - 5000 ml, e.g. of 500-2000 ml. The composition may be packed in the form of a single dose packaging (unit dose), or in a multidose container. Suitable packaging material is plastics, such as high-density polyethylene, HDPE. In one embodiment, the liquid composition is contained in a HDPE plastic container.

[0030] The claimed liquid composition provides a high concentration of the menthol as a stable solution. Studies of the applicant confirm that the composition is stable over time, typically of at least 24 months. Further studies now performed by the applicant document stability of the liquid menthol composition of at least 36 months, which is comparable with that of solid menthol flakes.

[0031] The composition of the invention is provided primarily for use in anesthesia of farmed fish, which may be food producing fish for human consumption. The disclosed composition is useful for farmed fish wherein this is from the group of bony fish (teleost), particularly for teleost fish species that are farmed commercially, and particularly for salmonid species. The fish is e.g. selected from the group comprising the Salmonids like Atlantic salmon (Salmo salarL), brown trout (Salmo trutta), Steelhead / Rainbow trout (Onchorhyncus mykiss), Chinook salmon (Oncorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), and Pacific salmonid fishes (Oncorhynchus s.p.p.). In a preferred embodiment, the composition is for use in anesthetic treatment of Atlantic salmon. In another embodiment, the fish is from a non-salmonid fish group, such as tilapia, catfish, sea bass or sea bream.

[0032] In another aspect, the invention relates to a method for preparing the composition of the first aspect, the method comprising the step of mixing an anesthetic agent, such as preferably a menthol, with the at least one excipient. The components of the composition are mixed, preferably by stirring, to completely solve the anesthetic agent. Optionally, the mixing is under gentle heating. When the composition has been prepared this is filled into suitable containers.

[0033] In further aspects, the invention relates to anesthetic compositions according to the first aspect for use in anesthesia of fish, and to methods for anesthesia of fish using the composition of the first aspect. Accordingly, the invention provides a composition for use in an anesthetic treatment method of fish, the use or method comprises a step of adding the liquid composition comprising an anesthetic agent selected from the group of menthols in a concentration of at least 20 weight% of the composition and at least one excipient comprising a propylene glycol, to a container comprising fish. In one embodiment, the composition for use comprises: i) 20-70% by weight of an anesthetic agent selected from the group of menthols; and ii) 30-80% by weight of a liquid vehicle comprising at least one excipient, wherein the at least one excipient comprises a propylene glycol.In one embodiment, the composition for use further comprises a further excipient, such as in a weight% of 0.1-20, wherein this further excipient is selected from nonionic surfactants, e.g. from the group of polysorbates and ethoxylated emulsifying agents, and preferably being macrogolglycerol hydroxystearate, as disclosed for the first aspect. The features disclosed for the first aspect apply also for aspects related to use of the composition in an anesthetic treatment method of fish.

[0034] In one embodiment, the use comprises a sedation step, such as a preanesthetic sedation step, wherein a low sedative dosage of the anesthetic composition is added to a container comprising the fish. Suitable dosages for such a sedation are 2-20 mg menthol / liter water, such as about 5 mg menthol / liter water. Accordingly, about 5-15 grams of the liquid composition should be added per 1000 liter of water for sedation. In this embodiment, after a minimum of about 10 minutes permitting the fish to be sedated it is then either:-moved to a tank containing a menthol in anesthetic doses, such as e.g. 80 to 320 mg / l, and after a minimum of 5 minutes euthanized by cutting of the gill arches, or-anesthetized using mechanical or electrical stunning, immediately followed by euthanasia by cutting of the gill arches, or-handled for procedures like crowding, treatments and transport.When the use comprises a first sedation step, followed by full anesthesia in a tank containing a menthol in anesthetic doses, the full anesthesia is obtained by adding about 160-480 grams of the liquid composition per 1000 liter water.

[0035] In another embodiment, the use comprises a direct anesthesia of fish and a higher anesthetic dosage of the anesthetic composition is added to a container comprising the fish. Suitable dosages are 80 to 320 mg menthol / liter water, such as about 160 mg menthol per liter water. Accordingly, about 160-480 grams, such as e.g. about 320 grams of the liquid composition should be added per 1000 liter water for full anesthesia. After a minimum of 5 minutes the fish may be euthanized by cutting of the gill arches.

[0036] It is to be understood that every embodiment of the disclosure can optionally be combined with any one or more of the other embodiments described herein. Elements disclosed for one aspect also apply for other aspects, hence the details provided for the composition also apply for the method or use when relevant.

[0037] It is to be understood that each component, compound, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, or parameter disclosed herein. It is further to be understood that each amount / value or range of amounts / values for each component, compound, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount / value or range of amounts / values disclosed for any other component(s), compound(s), or parameter(s) disclosed herein, and that any combination of amounts / values or ranges of amounts / values for two or more component(s), compound(s), or parameter(s) disclosed herein are thus also disclosed in combination with each other for the purposes of this description. Any and all features described herein, and combinations of such features, are included within the scope of the present invention provided that the features are not mutually inconsistent.

[0038] ExamplesExample 1: Preparation of anesthetic composition in the form of a liquid formulation of menthol40% w / w of 1 ,2-propylene glycol (as Kollisolv® PG) and 10% w / w macrogolglycerol hydroxystearate (as Kolliphor® RH 40) were weighed and blended forming a liquid vehicle. Levomenthol 50% w / w was added to the liquid vehicle and simultaneously mixed. The components were mixed until a clear, transparent and homogenous solution was achieved.The viscosity of the composition was measured at 20 °C, 15 °C and 5.9 °C by the method according to Ph. Eur. 11thEd, 01 / 2019:20209 Capillary Viscometer Method, with the following results:Kinematic viscosity, 20 °C: 93 mm2 / s2Dynamic viscosity, 20 °C: 90 mm2 / s2Kinematic viscosity, 15 °C: 137 mm2 / s2Dynamic viscosity, 15 °C: 131 mm2 / s2Kinematic viscosity, 5.9 °C: 283 mm2 / s2Dynamic viscosity, 5.9 °C: 273 mm2 / s2The liquid formulated composition was filled on HDPE containers. The stability of the composition has been monitored and inspected over time, showing stability of at least 24 months. No signs of precipitation are shown.The formulation of the example is named “Calmenthol vet. 500 mg / g”.Example 2: Stages of anesthesia on Atlantic salmonStudies have been performed by the applicant to document the effect of the claimed composition.The composition of Example 1 was used in the studies. The composition was tested on Atlantic salmon in different water temperatures, 5 °C and 15°C, at different dosages, and compared with existing anesthetics (Agui-S (isoeugenol 540 g / l), and Tricaine mesylate 1000 mg / g).Reference is made to Figures 1-4 attached, wherein stages of sedation versus time are shown in Figures 1 and 2, and stages of anesthesia and induction time areshown in Figures 3 and 4. The Y-axes show the sedation and anesthesia stages divided into the following:Stage 0: AlertStage I: Light sedationStage II: ExcitationStage III: Plane 1 Light anesthesiaStage III: Plane 2 - surgical anesthesiaStage III: Plane 3 - deep narcosisStage IV: Imminent death - MoribundIn the study of Figure 1 , the average sedative effect of Calmenthol (500 mg menthol / g composition) on Atlantic salmon (n=10) in dosages of 3.10 mg menthol per liter water and 5.15 mg menthol per liter water, respectively, was studied. The fish were exposed to an anesthetic bath for 30 minutes at 5°C. For comparison, a study using Aqui-S (2.48 mg isoeugenol / l) was conducted.It was found that for the given concentration (500 mg menthol / g) the dosage of 5.15 mg menthol per liter water is appropriate.Figure 2 is similar to Figure 1 , but shows the results from the study in water holding 15 °C.Stages of anesthesia and induction time is shown in Figures 3 and 4, from studies in water at 5 °C and 15 °C, respectively. Dosages of 82.7 mg / l and 165.3 mg / l Calmenthol and TMS (100 mg / l) were used. In these studies, the fish was exposed until surgical anesthesia was reached, then transferred to fresh water for recovery.In conclusion, the formulated liquid menthol composition was found efficient for both sedation and anesthesia, and both in water holding 5 °C and 15 °C.

Claims

Claims1. An anesthetic liquid composition comprising:20-70% by weight of an anesthetic agent selected from the group of menthols;20-60% by weight of an excipient being a propylene glycol;0.1-20% by weight of a further excipient selected from non-ionic surfactants from the group of polysorbates and ethoxylated emulsifying agents.

2. The liquid composition of claim 1 wherein the menthol is L-menthol.

3. The liquid composition of claim 1 or 2 wherein the propylene glycol is 1 ,2- propanediol.

4. The liquid composition of any of the preceding claims, wherein the further excipient is a non-ionic surfactant selected from the group of Polysorbates 20, 40, 60 and 80 and ethoxylated castor oil.

5. The liquid composition of any of the preceding claims, wherein the composition comprises a further excipient being the ethoxylated castor oil macrogolglycerol hydroxystearate.

7. The liquid composition of any of the preceding claims, wherein the composition comprises40-55 weight% of a menthol,35-45 weight% of a propylene glycol, and5-15 weight% of macrogolglycerol hydroxystearate.

8. The liquid composition of any of the preceding claims provided in a container of 10-10 000 ml.

9. An anesthetic liquid composition for use in anesthetic treatment of fish, the use comprises a step of adding the liquid composition according to any of the claims 1 to 8, to a water container comprising the fish.

10. An anesthetic liquid composition for use according to claim 9, wherein the composition is added to the water container comprising the fish in a dosage of 2-320 mg menthol / liter water.11 . The liquid composition for use according to claim 9 or 10, wherein the use comprises a sedation step with a sedative dosage of 2-20 mg menthol / liter water of the liquid composition.

12. The liquid composition for use according to claim 11 , wherein the fish is moved to a tank containing a menthol in anesthetic doses of 80 to 320 mg / l, after a minimum of about 10 minutes has permitted the fish to be sedated.

13. The liquid composition for use according to claim 9 or 10, wherein the use comprises a direct anesthesia of the fish with a dosage of 80-320 mg menthol / liter water of the liquid composition.

14. The liquid composition for use according to claim 9, wherein the fish is selected from fish species selected from the group of Salmonids Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta), Steelhead / Rainbow trout (Onchorhyncus mykiss), Chinook salmon (Oncorhynchus tshawytscha), Coho salmon (Oncorhynchus kisutch), and Pacific salmonid fishes (Oncorhynchus s.p.p.), and from the nonsalmonid fish group of tilapia, catfish, sea bass and sea bream.

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