WO1999024028A1 - Chlorphenesin as preservative - Google Patents

Abstract

The invention concerns the determination of a range of efficient concentrations of chlorphenesin in the presence or not of intervening substances. Said range is between 0.100 and 0.600 microgram of chlorphenesin per millimetre in final concentration ensuring prevention against contamination by micro-organisms. The demonstration of efficacy involves the concept of the most probable number (MPN) according to Mc Grady for heap micro-organisms such as S aureus and A: niger. Said efficient chlorphenesin concentrations are used for preventing contamination of dermo-cosmetic or pharmaceutical products such as orally administered solutes, vaccines, eye-potions, vaginal suppositories and the like.

Description

 Chlorophenesin preservative

The present invention relates to the prevention of contamination by microorganisms in oral and injectable solutions, in vaccines with a single antigenic component (monovalent vaccines) or with several components (combined or polyvalent vaccines), in sera and in products cosmetics. This prevention of contamination is, up to now, assumed or not by preservatives with poor efficacy (Phenoxy-ethanol, Metacresol, Phenol, Esters p.arahydroxybenzoic ...) or neurotoxic and neurocumulative (mercurial derivatives type Merthiolate ... ).

CHLORPHENESINE or 3p-Chlorphenoxypropane-1,2 diol or p-chlorphenyl-glycerol ether (MW = 202.6), used alone or in combination with another compatible preservative, overcomes this drawback.

This study has two stages:

- Determine an area of effective concentrations of CHLORPHENESINE.

- Demonstrate its effectiveness according to the requirements of the European and British Pharmacopoeias.

Determination of effective concentrations of CHLORPHENESIN

The effective concentration of chlorphenesin is the minimum bactericidal (C.M.B.) and fungicidal (C.M.F.) concentration

1.1. Microorganisms tested: The microorganisms used in determining the effective concentration of chlorphenesin are referenced A.T.C.C. (American Type Culture Collection):

- Escherichia coli ATCC 8739

- Pseudomonas aeruginosa ATCC 9027

- Staphylococcus aureus ATCC 6538 - Apergillus niger ATCC 16404

- Candida albicans ATCC 10231

1.2. Inoculum:

The microorganisms tested must be in exponential growth phase and in a liquid medium such as Mueller-Hinton Broth (b.M.H.) or Trypto-Casein-Soy Broth (b.T.C.S.) type.

The inoculum for the determination of C.M.B. and C.M.F. should be verified by:

REMFLACEMEN FINGER RULE 26 - Counting in agar medium Mueller-Hinton agar (gMH) or agar type TCS (gTCS) for E.coli, Ps.aeruginosa and C.albicans.

- Determination of the Most Probable Number (N.P.P.) in b.M.H. or b.T.C.S. according to the tables of Me GRADY for S.aureus and A.niger. Indeed, these are in .amas difficult to dissociate and cannot consequently be numbered in a conventional manner. 1.3. Minimum Concentration Inhihitrice fC.M.I - Minimum Concentration Bactericidal (C.M.B.) or Fungicide (C.M.F:

The determination of the C.M.I. is performed on a 96-well plate in a liquid nutrient medium type b.M.H. or type b.T.C.S. Chloφhenesin is diluted to the maximum of its solubility in b.M.H. or b.T.C.S. at

0.6% (W / V) at 20-25 ° C.

The final concentrations of Chlorphenesine tested follow a geometric progression of reason 2 with an initial concentration at 0.0125 microgram / ml and the highest at 0.400 microgram / ml. An inoculum control without chlorphenesin and the maximum concentration of 0.600 micrograms of chlorphenesin / ml are also tested.

The microorganism inoculum, from 1.10 ^ to 5.10 ^S viable units per 100 microliters well, must be in a negligible volume less than or equal to 5 microliters.

The plate, covered with a sterile cellophane sheet to avoid evaporation, is then incubated at 30-35 ° C for bacteria and at 20-25 ° C for yeast and mold.

Reading the C.M.I. is carried out after 24 h of incubation for bacteria and after 48 h for yeast and mold.

After recording the MIC, a subculturing of 100 micro liters of each of the wells with no apparent growth per 10 ml tube of bMH or bTCS allows, after incubation under the same conditions described for the MIC, to determine the CMB or the CMF The results are summarized in the table below: Table N ° 1: MIC and CMB or CMF of chlorphenesin

The effective concentration of chlorphenesin used as a preservative is therefore 0.400 micrograms / ml. The uncertainty being of one to two dilutions of difference, the zone of effective concentrations of chloφhenesine is therefore the interval 0.100 microgram / ml-0.600 microgram / ml; this latter concentration being the maximum solubility at room temperature 20-25 ° C.

11 / Efficacy of chloφhenesine as a preservative according to the requirements of the European and British Pharmacopoeias II.1. Principle:

The test consists of: - artificial contamination of an aqueous solution containing 10% (P / N) of any interfering substance: protein, lipid, carbohydrate using an inoculum of microorganisms (minimum 1.10 / ml ), in the presence of 0.400 microgram of chloφhenesin in final concentration per milliliter.

- maintaining the inoculated preparation at a temperature of 20-25 ° C and in the dark. - taking samples from the containers, at specified time intervals.

- the enumeration of microorganisms in the samples thus taken. II.2. European Pharmacopoeia criteria

II.3. Criteria of the British Pharmacopoeia:

II.4. Results: Note: - The inocula of E.coli, Ps.aéruginosa and C.albicans are verified by counting on Mueller-Hinton agar or T.C.S. On the other hand, those of S. aureus and A. niger are appreciated by their Most Probable Number according to Me GRADY in Mueller Hinton broth or in T.C.S.

- Any survivors of chloφhénésine are counted taking into account the residual content of chloφhénésine which must be, in the culture medium, less than or equal to

1/4 of the MIC for each microorganism. Table: Anti-microorganism activity of chloφhenesin at 0.40% W / V or 0.400 micrograms / ml. Logarythmic reduction / Peptone control at time T.

111 / Conclusion:

Chloφhenesin is effective as a preservative.

Claims

1) CHLORPHENESINE or 3p. CHLORPHENOXYPROPANE-1,2 DIOL used as a preservative. 2) CHLORPHENESINE according to claim 1 used at concentration ranges from 0.100 to 0.600 microgram / ml in the absence or in the presence of substances which may be interfering. Examples: protein, fat, carbohydrate. 3) Use of CHLORPHENESINE as a preservative in the cosmetic and pharmaceutical industries, for human or veterinary use. Examples: - Dermo-Cosmetics: Lip stick, shampoo, make-up remover, cream for facial or hand care, anti-acne cream or lotion, medico-surgical liquid soap, etc. - Pharmaceutical: Vaccines, serums, cream, ointment, suppository, ovum, eye drops, nasal solution, mouthwash, syrup, capsule, etc.