ICARIDIN HYDROXYETHYL ISOBUTYL PIPERIDINE CARBOXYLATE

Transcription

1 ICARIDIN HYDROXYETHYL ISOBUTYL PIPERIDINE CARBOXYLATE INFORMATION Common names Hydroxyethyl Isobutyl Piperidine Carboxylate (INCI), icaridin (WHO INN) Chemical name CA: 1- piperidinecarboxylic acid 2-(2-hydroxyethyl)-1- methylpropylester CAS Registry number Eilincs number CIPAC number 668 Structural formula N OH O O Empirical formula C 12 H 23 NO 3 Relative molecular mass Page 1 of 15

2 HYDROXYETHYL ISOBUTYL PIPERIDINE CARBOXYLATE Interim specification: WHO/IS/TC/668/2001 This specification, which is PART ONE of this publication, is based on an evaluation of data submitted by the manufacturer whose name is listed in the evaluation report (WHO/668/2001). It should be applicable to relevant products of this manufacturer but it is not an endorsement of those products, nor a guarantee that they comply with the specification. The specification may not be appropriate for the products of other manufacturers. The evaluation report (WHO/668/2001), as PART TWO, forms an integral part of this publication. 1 Description (Note 1) The material shall consist of Hydroxyethyl Isobutyl Piperidine Carboxylate together with related manufacturing impurities, in the form of a colourless and nearly odourless liquid, free from visible extraneous matter and added modifying agents 2 Active ingredient 2.1 ldentity tests (Note 1) The active ingredient shall comply with an identity test and, where the identity remains in doubt, shall comply with at least one additional test. 2.2 Hydroxyethyl Isobutyl Piperidine Carboxylate content (Note 1) The Hydroxyethyl Isobutyl Piperidine Carboxylate content shall be declared (not less than 970 g/kg) and, when determined, the mean measured content shall not be lower than the declared minimum content. 3 Relevant impurities 3.1 sec-butyl chloroformate (Notes 1.1 and 2) Maximum: 1 g/kg Note 1 Methods for sampling and analysis 1 Sampling. Thoroughly mix the contents of the container, remove an aliquot (not less than 50 ml) and place it in a clean colourless glass tube fitted with a ground glass stopper. Characteristics described by the specification should be assessed from this aliquot. 2 Identity tests 2.1 Gas Chromatography. Use the method described below. The retention time of Hydroxyethyl Isobutyl Piperidine Carboxylate, relative to internal standard, in the sample should not deviate by more than ±1.5% from that of the calibration solution. 2.2 IR (spectrum attached) 2.3 MS (spectrum attached) 3 Determination of Hydroxyethyl Isobutyl Piperidine Carboxylate content Page 2 of 15

3 OUTLINE OF METHOD Hydroxyethyl Isobutyl Piperidine Carboxylate is dissolved in dichloromethane, separated from other components by gas chromatography and quantified using a flame ionization detector and tetradecane internal standard. With appropriate calibration, this method may also be used to determine the content of relevant impurities at or above 0.5 g/kg. APPARATUS Gas chromatograph with flame ionisation detector (FID) and electronic data capture and integration. Capillary column, 30m length, 0.32 mm i.d., fused silica with DB-5 coating, film thickness 0.25 µm. REAGENTS Dichloromethane Tetradecane Internal standard solution: dissolve 500mg tetradecane in dichloromethane and make up to 20 ml. Hydroxyethyl Isobutyl Piperidine Carboxylate, reference standard Calibration solution: Weigh (to the nearest 0.1 mg) approximately100 mg Hydroxyethyl Isobutyl Piperidine Carboxylate, dissolve in 3 ml dichloromethane, add 1ml internal standard solution. PROCEDURE (a) Operating conditions (typical) Carrier Gas (Helium) approx. 1.5 ml/min Split flow 40 ml/min (1:28) Hydrogen 30 ml/min Air 300 ml/min Injector temperature 150oC Column oven temperatures initial programme rate final Detector temperature 300 o C Retention times 50 C (held for 2 min) 10 o C/min 300 o C (held for 10 min) Hydroxyethyl Isobutyl Piperidine Carboxylate 16.2 min tetradecane 13.5 min (b) System suitability test The Hydroxyethyl Isobutyl Piperidine Carboxylate may contain small amounts of ± hexahydro -1H, 3H- pyridol [1,2-c] [1,3]oxazin-1- one ( cyclocarbamate ). Ensure that the system provides a minimum of baseline resolution of tetradecane, cyclocarbamate and Hydroxyethyl Isobutyl Piperidine Carboxylate and that no peak in the sample coelutes with tetradecane. If the method is to be used to determine the content of relevant impurities, similar criteria should be adopted to assess chromatographic separation. Ensure that detector response to tetradecane and Hydroxyethyl Isobutyl Piperidine Carboxylate is linear at the concentrations to be measured, by injecting quantities in the range % of the nominal concentration. Modify the typical operating conditions to comply with these requirements, if necessary. (c) Preparation of sample Weigh (to the nearest 0.1 mg) approximately 100 mg of sample and dissolve in 3 ml dichloromethane. Add 1.0 ml Internal Standard. (d) Determination Inject 1 µl portions of the calibration solution until the response factor for successive injections differs by less than ±1%. Then make duplicate injections of the sample solution followed by another injection of the calibration solution. (e) Calculation Calculation of the response factor Page 3 of 15

4 W Oi * F 02 * r fi = W O2 * F Oi * 1000 Where: fi = response factor of component i F Oi = peak area of component i F 02 = peak area of tetradecane W Oi = weight of component i taken (mg) W O 2 = weight of tetradecane taken (mg) r = purity of standard of component i in g/kg 1000 = conversion factor to g/kg Calculation of the content of the component i in the sample F i * W 2 * f i * 100 W i = W 1 * F 2 W i = content of component i in % F i = peak area of component i F 2 = peak area of tetradecane W 2 = weight of tetradecane taken (mg) f i = response factor of component i W 1 = weight of sample taken (mg) 100 = conversion factor to % Note 2 Determination of sec-butyl chloroformate content OUTLINE OF METHOD A sample is dissolved in dry dichloromethane and analyzed by capillary gas chromatography and sec-butyl chloroformate is quantified using a flame ionization detector. Use of dried dichloromethane is essential to avoid hydrolysis of the sec-butyl chloroformate. Calibration may be by external standardization or by internal standardization using tetradecane as the internal standard. Internal standardization may be helpful where high precision is required. APPARATUS Gas chromatograph with flame ionisation detector (FID) and electronic data capture and integration. Capillary column, 30m length, 0.25 mm i.d., fused silica with cross-linked 100% dimethyl polysiloxane coating, film thickness 0.25 µm. REAGENTS Dichloromethane (Merck LiChrosolv Nr , or equivalent, dried over molecular sieve Bayer Zeolith SE 134, or equivalent). Tetradecane Internal standard solution: accurately weigh about 500mg tetradecane, dissolve in dichloromethane and make up to 500 ml in a flask. sec-butyl chloroformate, reference standard Calibration solutions: accurately weigh approximately10 mg sec-butyl chloroformate into a 10 ml flask and dilute to volume with dichloromethane. Transfer 1 ml of this solution to a 10 ml flask and add 9 ml internal standard solution if required, otherwise make to volume with dichloromethane. PROCEDURE Page 4 of 15

5 (a) Operating conditions (typical) Carrier Gas (hydrogen) approx. 1.2 ml/min Split flow 1:10 Hydrogen 30 ml/min Air 300 ml/min Injector temperature 150oC Column oven temperatures initial 50 C (held for 2 min) programme rate 10 o C/min final 260 o C Detector temperature 320 o C Retention times Hydroxyethyl Isobutyl Piperidine Carboxylate16.3 min sec-butyl chloroformate 3.9 min tetradecane 13.0 min (b) System suitability test The Hydroxyethyl Isobutyl Piperidine Carboxylate may contain small amounts of ± hexahydro -1H, 3H- pyridol [1,2-c] [1,3]oxazin-1- one ( cyclocarbamate ). Ensure that the system provides a minimum of baseline resolution of tetradecance (if used), cyclocarbamate and Hydroxyethyl Isobutyl Piperidine Carboxylate and that no peak in the sample co-elutes with tetradecane. Ensure that detector response to tetradecane (if used) and sec-butyl chloroformate is linear at the concentrations to be measured, by injecting quantities in the range % of the nominal concentration. Modify the typical operating conditions to comply with these requirements, if necessary. (c) Preparation of sample Accurately weigh approximately 500 mg of sample and 10 mg tetradecane (if required), dissolve in dichloromethane and make to 10 ml final volume. (d) Determination Inject 2 µl portions of the calibration solution and sample solution until the response factor for successive injections differs by less than ±1%. Then make duplicate injections of the sample solution followed by another injection of the calibration solution. (f) Calculation Calculation of the response factor of sec-butyl chloroformate (SBCF), fi (weight IS )(g/kg purity IS )(peak area sbcf REF ) fi = (weight sbcf REF )(g/kg purity sbcf REF )(peak area IS ) Calculation of the content of sec-butyl chloroformate in the sample (weight IS )(g/kg purity IS )(peak area sbcf REF ) fi = (weight SAMPLE )(peak area IS ) * fi Page 5 of 15

6 HYDROXYETHYL ISOBUTYL PIPERIDINE CARBOXYLATE (WHO/668/2001) EVALUATION REPORT Explanation The data on Hydroxyethyl Isobutyl Piperidine Carboxylate (also known as Bayrepel and icaridin) were evaluated in support of a new WHO specification. This active ingredient is under patent in Denmark, Norway, Finland, Hungary, South Africa, Turkey, Thailand, Philippines, Japan, Brazil, Venezuela, Australia, New Zealand and Mexico until 2008; in the USA until 2009; in Canada until 2011; and in Argentina until The European Patent is valid until Hydroxyethyl Isobutyl Piperidine Carboxylate has been evaluated for efficacy by the WHOPES programme (ref. 1). It has also been evaluated by the WHO/PCS (ref. 1). The draft specification and supporting data were provided by Bayer AG, Germany, in Uses Hydroxyethyl Isobutyl Piperidine Carboxylate is an insect repellent for application to human or animal skin. It also shows repellent activity when applied on surfaces. It is used in public health applications to repel biting insects such as mosquitoes and biting flies (ref. 1). Identity INCI name: Hydroxyethyl Isobutyl Piperidine Carboxylate Chemical names: IUPAC: 1- methylpropyl 2- (2- hydroxyethyl)-1- piperidinecarboxylate CA: 1- piperidinecarboxylic acid 2-(2-hydroxyethyl)-1- methylpropylester CAS No: Elincs No: CIPAC No: 668 Synonyms: Bayrepel, KBR 3023, Propidine, Pikaridin, Icaridin (WHO INN) Structural formula: N OH O O Page 6 of 15

7 Molecular formula: C 12 H 23 NO 3 Relative molecular mass: Identity tests:gc (relative retention time), IR, MS (reference spectra deposited with WHO) Physico-chemical properties of Hydroxyethyl Isobutyl Piperidine Carboxylate TGAI (Table 1) Parameter Value(s) and conditions Purity % Method reference Vapour pressure: 3.4x10E-2 PA at 20 o C 5.9x10E-2 PA at 25 o C 97% OECD 104 Freezing point and thermal stability: Freezing point: -170 C Thermally stable at ambient temperatures under air DTA: Stable under air for up 160 C under nitrogen for up to 260 C No exothermic reaction until 400 C TGA: Weight loss starting 120 C 97% EU- Method A1 OECD 113 Solubility in water: 8.2 g/l at 20 C at ph 4-9: 8.6 g/l at 20 C in unbuffered water: 97% OECD 105 Solubility in organic solvents Octanol / water partition coefficient: Hydrolysis characteristics: Photolysis characteristics: Dissociation characteristics: log P ow 2.11 at 20 C unbuffered log P ow 2.23 at 20 C at ph 4-9: No hydrolysis products observed after 7 days at 50 C and after 30 days at 25 C at ph 5, 7 and 9 No absorption at UV and visible wavelengths No acidic or basic properties in aqueous solution 97% OECD % EPA Pesticide Assessment Guideline, Subdivision N, Section 161-1, % OECD 112 Page 7 of 15

8 Chemical composition and properties of the technical material (Table 2) Manufacturing process, maximum limits for impurities 1 g/kg, two series of 5 batch analysis data. Declared minimum Hydroxyethyl Isobutyl Piperidine Carboxylate content: Relevant impurities 1 g/kg and maximum limits for them: Relevant impurities < 1 g/kg and maximum limits for them: Confidential information supplied and held on file by WHO. Mass balances: and g/kg were accounted for as active ingredient and identified impurities, from analysis of two series of 5 batches. 970 g/kg None sec-butyl chloroformate, 1 g/kg Stabilisers or other additives and maximum None limits for them: Melting or boiling temperature range Melting point: below -170 C Boiling point: 296 C (calculated from vapour pressure) No exothermic reaction until 400 C Page 8 of 15

9 Toxicological summaries Notes. (i) The proposer confirmed that the toxicological and ecotoxicological data included in the summary below were derived from Hydroxyethyl Isobutyl Piperidine Carboxylate having impurity profiles similar to those referred to in Table 2. (ii) The conclusions expressed in the summary below are those of the proposer, unless otherwise specified. Table 3. Toxicology profile of the technical material, based on acute toxicity, irritation and sensitization. Species Test Duration and conditions or guideline adopted Rat, m oral, fasted OECD 401 Result LD 50 = 2236 mg/kg bw (approximate), NOEL: 100 mg/kg bw Rat, m oral, non fasted OECD 401, EPA-FIFRA 81-1 LD 50 = 4743 mg/kg bw (approximate), NOEL: 100 mg/kg bw Rat, m dermal OECD 402 LD 50 > 5000 mg/kg bw, NOEL: 5000 mg/kg bw Rat, m/f dermal EPA-FIFRA 81-2, US-EPA-TSCA No , OECD 402, JMAFF 59 NohSan No Rat, m inhalation Exposition: 1x4 hours, OECD 403 Rabbit, m skin irritation OECD 404 non irritant LD 50 > 2000 mg/kg bw, NOEL: 5000 mg/kg bw LC 50 > 4364 mg/m 3 air, NOEL: 2153 mg/m 3 air Rabbit, m eye irritation OECD 405 slightly irritant Guinea pig, m skin sensitization OECD 406, EU- 84/449/EEC non sensitizing Page 9 of 15

10 Table 4. Toxicology profile of the technical material based on repeated administration (sub-acute to chronic) Species Test Duration and conditions or guideline adopted Rat Sub-chronic dermal 13 weeks EPA FIFRA 82-3, OECD 411, JMAFF 59 NohSan No Dog Rat Chronic dermal (started as sub-chronic study) Combined chronic toxicity/carcinogenicity dermal 1 year EPA FIFRA 83-1, 82-2, US EPA TSCA No , OECD 453, JMAFF 59 NohSan No years EPA FIFRA No. 83-5, OECD 453, TSCA , JMAFF 59 NohSan No Mouse Oncogenicity dermal 13 months EPA FIFRA No. 83-2, OECD 451, TSCA , JMAFF 59 NohSan No Rat 2-generation dermal 2 generations EPA FIFRA No. 83-4, OECD 416, TSCA , JMAFF 59 NohSan No. 4200, EU 87/302/EEC Rat Rabbit Rat Teratogenicity and developmental toxicity Teratogenicity and developmental toxicity Sub-chronic neurotoxicity dermal GD 0-20 EPA FIFRA No. 83-3, OECD 414, TSCA , JMAFF 59 NohSan No. 4200, EU 87/302/EEC GD 0-20 EPA FIFRA No. 83-3, OECD 414, JMAFF 59 NohSan No. 4200, EU 87/302/EEC 13 weeks US EPA FIFRA 82-5(b) Result LOEL = 500mg/kg bw/d NOEL = 200mg/kg bw/d LOEL = >200mg/kg bw/d NOEL = 200mg/kg bw/d LOEL = 200mg/kg bw/d NOEL = 100mg/kg bw/d LOEL = > 200mg/kg bw/d NOEL = 200mg/kg bw/d LOEL = > 200mg/kg bw/d NOEL = 200mg/kg bw/d LOEL = 400mg/kg bw/d NOEL = 200mg/kg bw/d LOEL = 200mg/kg bw/d NOEL = 100mg/kg bw/d LOEL = > 200mg/kg bw/d NOEL = 200mg/kg bw Page 10 of 15

11 Table 5. Mutagenicity profile of the technical material based on in vitro and in vivo tests Species Test Conditions Result Salmonella typhimurium hamster ovarian cells Salmonella/microsome test in vitro CHO-HGPRT Assay in vitro OECD 471, EPA FIFRA 84-2 EU 87/302/EEC, OECD 475, EPA FIFRA PB V79 cells V79/HPRT-test in vitro EU 88/302/EEC, OECD 476, EPA FIFRA OPPTS Chinese hamster ovary cells mouse rat primary hepatocytes Chromosome aberration test cytogenetics in vitro Micronucleus test in vivo unscheduled DNA synthesis assay in vitro EU 79/831/EEC, OECD 473, EPA FIFRA 84-2 EU 92/69/EEC, OECD 474, EPA FIFRA PB EU 87/302/EEC, OECD 482, EPA FIFRA PB negative negative negative slightly positive within the toxic range negative negative Page 11 of 15

12 Table 6. Ecotoxicology profile of the technical material Species Test Duration and conditions Result Daphnia magna (water flea)] Bacteria (mixed population) Oncorrhynchus mykiss (rainbow trout) Brachydanio rerio Hamilton Buchanan (zebra danio) Scenedesmus subspicatus (green alga) acute toxicity 48h, static, 20+/- 1 0 C OECD 202 EPA- FIFRA 72-2 toxicity 88/302/EEC, EC L 133, Part 3 (corresponds in most parts to OECD 209) acute toxicity, 96 h, static OECD 203 EPA- FIFRA 72-1 EG L 383 A/163 EC 50 : calculation not possible 103 mg/ l = NOEC (highest test conc.) EC 50 : 1110 mg/l no effect on the bacterial population LC 50 = 173 mg/l NOLEC= 85.6 mg/l bioaccumulation OECD 305 no potential for bioaccumulation in fish effect on growth 72h, 23+/-2 0 C EEC Directive 79/831/E Rev.Vs.383A/179 ISO 8692 (1989) OECD 201 (1984) EC 50 = 89.2 mg/l NOEC = 56 mg/l Earthworm - - Not applicable due to use pattern Apis mellifera (honey bee) Colinus virgianus (bobwhite quail) - - Not applicable due to use pattern Five-day dietary toxicology EPA- FIFRA 71-2 OECD 205 LC 50 = >5000 ppm a.i. in diet NOEC= 5000 ppm a.i. in diet The hazards and risks associated with Hydroxyethyl Isobutyl Piperidine Carboxylate were evaluated by WHO/PCS, as part of this review and also as part of the WHOPES evaluation of efficacy. The WHO/PCS evaluation concluded that the intended uses of the material posed no unacceptable risks to users, treated animals or to the environment. WHO/PCS has proposed Class slightly hazardous for Hydroxyethyl Isobutyl Piperidine Carboxylate, pending final approval of this classification in Formulations The main formulation types available are alcohol-based liquids, aerosols, emulsions (body lotions), sticks (such as deodorant sticks). These formulations are registered and sold in many countries throughout the world. Methods of analysis and testing The analytical method for determination of the quantity and identity of the active ingredient is based on capillary GC with FID and internal standardisation with tetradecane. The same method may be used to determine the presence of the Page 12 of 15

13 relevant impurities when they occur at >0.5 g/kg. Alternatively, separate methods may be used, for which peer validation are available. The identity of the active ingredient may established by comparing GC relative retention time, IR or MS spectra with those of an authentic standard. Copies of the IR spectrum and mass spectrum are held by WHO. The methods for determination of impurities were based on GC with FID. The material accountability study was performed according to the US Environmental Protection Agency s, Pesticide Assessment Guidelines, Subd. D; Series 62. Physical properties Test methods used to determine the physical properties of technical active ingredient were OECD, EPA, EU, Ph. Eur., while those for the formulations were OECD, EPA, EU, Ph. Eur or DIN/ ISO. Containers and packaging The active ingredient and its formulations can be stored in containers of polyethylene or high density polyethylene. Expression of the active ingredient The active ingredient is expressed as Hydroxyethyl Isobutyl Piperidine Carboxylate. Appraisal Hydroxyethyl Isobutyl Piperidine Carboxylate (Bayrepel ) is a patented active ingredient that has not previously been the subject of WHO specifications. Its INCI common name is less convenient for general use than the WHO International Non-proprietary Name, icaridin. The molecule has two chiral centres and exists as two pairs of diastereoisomers, which are not readily separated by gas chromatography. The compound is moderately soluble in water and very soluble in a wide range of organic solvents but it is not classed as fat soluble. It has no acidic or basic characteristics. Measurable hydrolysis does not occur in water at ph 5, 7 and 9 after 7 days at 50 C and after 30 days at 25 C. Photolysis is unlikely as it has no appreciable absorption at UV wavelengths. Confidential information on the manufacturing process, and the impurities present at or above 1 g/kg, was provided by the proposer. Mass balances were high, in the range g/kg in one series of 5 batches reported and g/kg in a second series of 5 batches reported. Hydroxyethyl Isobutyl Piperidine Carboxylate is not amenable to the most commonly used non-specific methods for the determination of purity of certified reference material. In the course of the second study, the original reference material was found to be slightly more pure than originally determined (989 g/kg instead of 981 g/kg). Mass balances were therefore presumably slightly under-estimated in the first study. The proposer originally suggested that 5 impurities (including water and another solvent) should be regarded as relevant impurities but agreed that there was no evidence to show that they had any adverse toxicological, ecotoxicological, Page 13 of 15

14 product stability, efficacy or taint/odour properties. The proposer s manufacturing specification requires that no impurity, other than the 2 solvents and 3 byproducts, should exceed 1 g/kg. There are no specific toxicological and ecotoxicological data available for the 3 by-products of synthesis identified. The minimum purity of the TC is high (970 g/kg) and the WHO/PCS opinion was that there was no evidence to suggest that the 3 by-products are more toxic than the active ingredient. The proposer noted that synthesis of Hydroxyethyl Isobutyl Piperidine Carboxylate has the potential to produce sec-butyl chloroformate and, hypothetically, sec-butyl carbonic anhydride (KBA 1542) as impurities. Both are reactive compounds and are easily hydrolysed. A report was provided (Diesing 1991) to show that they have strong skin sensitizing properties, although they do not sensitize skin to hydroxyethyl isobutyl piperidine carboxylate. In principle, both impurities can be considered as relevant. No other hazardous impurities, such as nitrosamines, are known to be associated with the synthesis of Hydroxyethyl Isobutyl Piperidine Carboxylate. The proposer stated that sec-butyl carbonic anhydride had never, in practice, been detected in the synthesis and manufacture of Hydroxyethyl Isobutyl Piperidine Carboxylate. The proposer observed that if, hypothetically, traces of sec-butyl carbonic anhydride were to be produced during synthesis, it would not survive the conditions employed for isolation of the active ingredient. It had not been necessary to optimise the manufacturing process to eliminate or minimise occurrence of this impurity. The analytical method used for the determination of other impurities (ref. 2) was not validated for the determination of sec-butyl carbonic anhydride. Nonetheless, sec-butyl carbonic anhydride is detectable by this method and validation data for the other impurities indicate that not detected may be interpreted as <0.1 g/kg. The toxicological data (ref. 3) available did not permit estimates of the LOAEL and NOAEL for sec-butyl carbonic anhydride. The opinion of WHO/PCS was that the level of sec-butyl carbonic anhydride in the TC should not be permitted to exceed 1 g/kg, because it is known that this level was not exceeded in the technical materials actually used for the toxicity testing. Given that there is no evidence to suggest that the impurity is likely to occur at detectable levels, a clause to limit its concentration appears unnecessary and would needlessly increase the cost of checking compliance with the specification. In future, if specifications are proposed for Hydroxyethyl Isobutyl Piperidine Carboxylate produced by other manufacturers, the possibility of sec-butyl carbonic anhydride occurring in their products should be considered, in case it is necessary to limit the concentration. The proposer stated that the manufacturing process had been designed to minimize the possibility of occurrence of sec-butyl chloroformate and that, in practice, this impurity is not detectable (i.e. < 0.05 g/kg). It is readily hydrolysed but the possibility of its presence is not hypothetical. Therefore this impurity is relevant and should be limited by an appropriate specification clause. The toxicological data available did not permit estimates of the LOAEL and NOAEL for sec-butyl chloroformate. The opinion of WHO/PCS was that the level of secbutyl chloroformate in the TC should not be permitted to exceed 1 g/kg, because it is known that this level was not exceeded in the technical materials actually Page 14 of 15

15 used for the toxicity testing. A concentration nominally 5 times higher than this did not induce a reaction in sensitized animals in a guinea pig maximization test. The proposer confirmed that technical grade Hydroxyethyl Isobutyl Piperidine Carboxylate, complying with this limit, showed no skin sensitization properties. The analytical method for the determination of sec-butyl chloroformate and various other impurities has been validated for linearity, precision, accuracy, specificity and limits of determination and detection. It is described in a Note to the specification. It has not been peer validated, which is required to support a full WHO specification. The accuracy and precision, as measured by recovery (a TC containing detectable levels of this impurity could not be obtained) at approximately 0.9 g/kg, was 62% with RSD 8%. The losses and hence relatively poor accuracy were ascribed to the high reactivity and ease of hydrolysis of the analyte. Specially dried solvent is required for the analytical method and it seems probable that the losses of sec-butyl chloroformate were due to reaction with small amounts of water present in the TC. Therefore the method is likely to be more accurate than the recovery data suggest and there should be no need to adjust the specified limit to take into account the apparent inaccuracy of the analytical method. The limit of quantification, estimated from the calibration data generated in the presence of pure Hydroxyethyl Isobutyl Piperidine Carboxylate (i,e, not the TC), was 0.1 g/kg. However, significant and rather variable losses were observed in recovery from the TC at 0.9 g/kg. Although these losses may have been due to water in the TC, rather than in the analytical method, there is no direct evidence of the overall accuracy and precision of the method at 0.1 g/kg. A limit of 1 g/kg, being the concentration which was not exceeded in the technical material actually used for toxicity testing, is acceptable to WHO/PCS and achievable analytically and it should therefore be used as the limit in the specification. The analytical method for determination of the active ingredient has not been validated by collaborative study, although the proposer agreed to organise such a study in support of a full specification. However, peer validation data were provided (ref. 4) to show that the method is robust for determination of the active ingredient content of the TC. Validation by collaborative study should precede adoption of the specification as full by WHO. The analytical method is described in a Note appended to the specification. The identity tests are based on GC relative retention time, IR and MS spectra and are fully satisfactory for the purpose. Physical and toxicological test methods followed internationally recognised protocols. Literature on efficacy of the active ingredient is beyond the scope of specifications and therefore was not accessed or considered for this evaluation. Recommendations The proposed specification should be adopted as an interim specification by WHO. To enable the interim specification to be considered for full status, the proposer should conduct a collaborative study of the method for determination of the active ingredient and a peer validation of the method for determination of secbutyl chloroformate. Page 15 of 15

16 If the specification is extended in future to include the products of other manufacturers, the evaluation must consider the need for a clause to limit the content of sec-butyl carbonic anhydride (KBA 1542). If the present proposer changes the manufacturing process such that detectable levels of this impurity could occur, the proposer must inform WHO so that the specification can be reviewed. References 1. World Health Organization (2001). Report of the fourth WHOPES Working Group meeting. Geneva, WHO document WHO/CDS/WHOPES/ Reubke K.-J. (2001). Validation of GC method , determination of impurities in technical active ingredient of KBR Bayer confidential report No. VB Diesing L. (1991). Components of KBR 3023: study for skin sensitising effect on guinea pigs. Bayer AG confidential report No Reubke K.-J. (2001). Validation of GC method , determination of KBR 3023 in technical active ingredient. Bayer confidential report No. VB Page 16 of 15