Opinion Of The Scientific Committee On Consumer Safety Benzisothiazoli P96 Sccs O 099
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SCCS/1482/12 Scientific Committee on Consumer Safety SCCS OPINION ON Benzisothiazolinone COLIPA n° P96 The SCCS adopted this opinion at its 15th plenary meeting of 26-27 June 2012 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ About the Scientific Committees Three independent non-food Scientific Committees provide the Commission with the scientific advice it needs when preparing policy and proposals relating to consumer safety, public health and the environment. The Committees also draw the Commission's attention to the new or emerging problems which may pose an actual or potential threat. They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) and are made up of external experts. In addition, the Commission relies upon the work of the European Food Safety Authority (EFSA), the European Medicines Agency (EMA), the European Centre for Disease prevention and Control (ECDC) and the European Chemicals Agency (ECHA). SCCS The Committee shall provide opinions on questions concerning all types of health and safety risks (notably chemical, biological, mechanical and other physical risks) of non-food consumer products (for example: cosmetic products and their ingredients, toys, textiles, clothing, personal care and household products such as detergents, etc.) and services (for example: tattooing, artificial sun tanning, etc.). Scientific Committee members Jürgen Angerer, Ulrike Bernauer, Claire Chambers, Qasim Chaudhry, Gisela Degen, Elsa Nielsen, Thomas Platzek, Suresh Chandra Rastogi, Vera Rogiers, Christophe Rousselle, Tore Sanner, Jan van Benthem, Jacqueline van Engelen, Maria Pilar Vinardell, Rosemary Waring, Ian R. White Contact European Commission Health & Consumers Directorate D: Health Systems and Products Unit D3 - Risk Assessment Office: B232 B-1049 Brussels Sanco-SCCS-Secretariat@ec.europa.eu © European Union, 2012 ISSN 1831-4767 ISBN 978-92-79-30762-1 Doi:10.2772/83092 ND-AQ-12-012-EN-N The opinions of the Scientific Committees present the views of the independent scientists who are members of the committees. They do not necessarily reflect the views of the European Commission. The opinions are published by the European Commission in their original language only. http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm 2 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ ACKNOWLEDGMENTS Prof. J. Angerer Dr. U. Bernauer Dr. C. Chambers Prof. G. Degen Dr. W. Lilienblum Dr. E. Nielsen Dr. S.C. Rastogi Prof. V. Rogiers Prof. T. Sanner Dr. J. van Engelen Prof. R. Waring Dr. I.R. White (rapporteur) (associate scientific advisor) (chairman) Keywords: SCCS, scientific opinion, preservative, 76/768/ECC, CAS 2634-33-5, EC 220-120-9 benzisothiazolinone, P96, directive Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on benzisothiazolinone, 26-27 June 2012 This opinion has been subject to a commenting period of four weeks after its initial publication. Comments received during this time have been considered by the SCCS and discussed in the subsequent plenary meeting. Where appropriate, the text of the relevant sections of the opinion has been modified or explanations have been added. In the cases where the SCCS after consideration and discussion of the comments, has decided to maintain its initial views, the opinion (or the section concerned) has remained unchanged. Revised opinions carry the date of revision. 3 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................... 3 1. BACKGROUND ............................................................................................. 5 2. TERMS OF REFERENCE.................................................................................. 5 3. OPINION..................................................................................................... 6 3.1. Chemical and Physical Specifications....................................................... 6 3.1.1. 3.1.2. 3.1.3. 3.1.4. 3.1.5. 3.1.6. 3.1.7. 3.1.8. 3.1.9. 3.2. Function 3.3. Chemical identity ........................................................................ 6 Physical form ............................................................................. 6 Molecular weight ........................................................................ 6 Purity, composition and substance codes........................................ 7 Impurities / accompanying contaminants ....................................... 7 Solubility ................................................................................... 7 Partition coefficient (Log Pow) ...................................................... 8 Additional physical and chemical specifications................................ 8 Homogeneity and Stability ........................................................... 8 and uses ................................................................................ 8 Toxicological Evaluation ........................................................................ 9 4. 3.3.1. Acute toxicity ............................................................................. 9 3.3.2 Irritation and corrosivity ............................................................ 10 3.3.3. Skin sensitisation...................................................................... 13 3.3.4. Dermal / percutaneous absorption............................................... 16 3.3.5. Repeated dose toxicity .............................................................. 18 3.3.6. Mutagenicity / Genotoxicity ........................................................ 20 3.3.7. Carcinogenicity......................................................................... 23 3.3.8. Reproductive toxicity................................................................. 23 3.3.9. Toxicokinetics .......................................................................... 25 3.3.10. Photo-induced toxicity ............................................................... 25 3.3.11. Human data ............................................................................. 25 3.3.12. Special investigations ................................................................ 26 3.3.13. Safety evaluation (including calculation of the MoS)....................... 26 3.3.14. Discussion ............................................................................... 26 CONCLUSION ............................................................................................ 28 5. MINORITY OPINION.................................................................................... 28 6. REFERENCES ............................................................................................. 29 4 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 1. BACKGROUND 1,2-Benzisothiazol-3(2H)-one (CAS No 2634-33-5, EC No 220-120-9) with the INCI name benzisothiazolinone is currently not listed in Annex VI of the Cosmetics Directive and therefore cannot be used as preservative. COLIPA has requested the inclusion of benzisothiazolinone in Annex VI in order to allow the use of benzisothiazolinone in cosmetic products. A first scientific opinion (SCCNFP/0811/04) was adopted 1 July 2004 by the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers with the following opinion: "The SCCNFP is of the opinion that the information submitted is insufficient to assess the safe use of benzisothiazolinone. Before any further consideration, the following information is required: * * percutaneous absorption study in accordance with the SCCNFP Notes of Guidance; reproduction toxicity data." The present submission II provides the data requested by this opinion. 2. TERMS OF REFERENCE 1. Does SCCS consider benzisothiazolinone safe when used as a preservative up to a maximum authorised concentration of 0.01% in cosmetic products, based on the provided data? 2. And/or does the SCCS have any scientific concern with regard to the use of benzisothiazolinone in cosmetic products? 5 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 3. OPINION 3.1. Chemical and Physical Specifications Benzisothiazolinone is listed in the EU Cosmetics Inventory, Section 1 with indicated function "antimicrobial". It is currently not regulated in the annexes of the Cosmetics Directive 76/768/EEC. 3.1.1. Chemical identity 3.1.1.1. Primary name and/or INCI name Benzisothiazolinone (INCI) 3.1.1.2. Chemical names 1,2-Benzisothiazol-3(2H)-one (IUPAC) 1,2-Benzisothiazol-3-one 1,2-Benzisothiazolin-3-one Benzo[a]isothiazol-3-one 3.1.1.3. Trade names and abbreviations BIT; Thor BIT; ACTIDE® BIT; Microcare® BIT; Nuosept BIT Technical; Promex BIT COLIPA P96 3.1.1.4. CAS / EC number CAS: EC: 2634-33-5 220-120-9 3.1.1.5. Structural formula 3.1.1.6. Empirical formula Formula: C7H5NOS 3.1.2. Physical form Off-white to yellowish solid 3.1.3. Molecular weight Molecular weight: 151.19 g/mol 6 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 3.1.4. Purity, composition and substance codes Substance code: Batches used: Purity: Loss on drying: Water content: Ash content: Sodium chloride: Lead: Mercury: Arsenic: Iron: / batch no 2001 014 74.02-84.02% w/w (84% corresponds to 15% water content) > 99% w/w on a dry basis / 15-29% w/w (for batch no 2001 014 spec., 20% max, found 15%) / < 0.1% w/w (for batch no 2001 014 spec., 0.2% max, found 0.02%) / / / / Impurities • 5-Chloro-1,2-benzisothiazolin-3(2H)-one: 0.15-0.22% w/w (for batch no 2001 014, specification 4 ppm max, found 3 ppm) CAS: 4337-43-3 EC: 224-385-1 emp. Formula: C7H4ClNOS • 2,2-Dichlorobisbenzamide: < 0.1% w/w (for batch no 2001 014, specification 0.5% max, found 0.03%) CAS: 2527-57-3 EC: 219-766-4 emp. Formula: C14H12N2O2S2 Residual solvents: 3.1.5. / Impurities / accompanying contaminants See 3.1.4. 3.1.6. Solubility Solubility in water: 1.1 g/l (0.11%) at 20 °C 6.0 g/l (0.60%) at 30 °C (Directive 92/69/EEC, A6) Effect of pH and temperature on solubility in water (OECD Guideline 105) at 10 °C and pH 4.8: 0.736 g/l at 20 °C and pH 4.8: 0.938 g/l 7 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ at 30 °C and pH 4.8: 1.198 g/l at 20 °C and pH 6.7: 1.288 g/l at 20 °C and pH 9.1: 1.651 g/l 3.1.7. Partition coefficient (Log Pow) Log Pow: 0.4 at 20 °C (OECD Guideline 117) Effect of pH and temperature on Log Pow (OECD Guideline 117 (HPLC)) Log Log Log Log Log Pow: Pow: Pow: Pow: Pow: 0.99 at 20 °C and pH 5 0.63 at 10 °C and at pH 7 0.70 at 20 °C and pH 7 0.76 at 30 °C and pH 7 – 0.90 at 20 °C and pH 9 Conclusions With increasing pH from 5 to 9, the Log Pow decreases very strongly. Only a slight increase of Log Pow is observed between 10 °C and 30 °C. 3.1.8. Additional physical and chemical specifications Melting point: Boiling point: Density: Vapour Pressure: pKa: Flash point: Viscosity: Refractive index: UV_Vis spectrum (200-800 nm): 3.1.9. 156.6 °C (Directive 92/69/EEC, A1) 327.6 °C (Directive 84/449/EEC, A2) 1.483 g/cm³ at 20 °C (OECD Guideline 109) 0.0000037 hPa at 25 °C (Directive 92/69/EEC, A4) 7.3 at 25 °C / / / / Homogeneity and Stability No data General Comments to physico-chemical characterisation No data on stability are provided 3.2. Function and uses Benzisothiazolinone is listed in the EU Cosmetics Inventory, Section 1 with indicated function "antimicrobial". It is currently not regulated in the annexes of the Cosmetics Directive 76/768/EEC. The submitted dossier requests a maximum level of 100 ppm as preservative in cosmetic products. This is greater than the level proposed in the earlier submission, which the applicant justifies with the availability of more recent studies, demonstration of a greater margin of safety. Aside from its use in cosmetic products, there are also other uses: Under the Biocide review programme for existing substances (Commission Regulation (EC) No 1451/2007), Benzisothiazolinone is examined for use in the following product types: 8 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Private area and public health area disinfectants and other biocidal products (2), In-can preservatives (6), Film preservatives (7), Fibre, leather, rubber and polymerised materials preservatives (9), Masonry preservatives (10), Preservatives for liquid-cooling and processing systems (11), Slimicides (12), Metalworking-fluid preservatives (13), Embalming and taxidermist fluids (22). Benzisothiazolinone is used as a slimicide in the manufacture of disposable powder-free polyvinyl chloride gloves. (Add. ref. 41) Benzisothiazolinone is widely used in industry as a preservative in water-based solutions such as pastes, paints and cutting oils. (Add. ref. 42, 43) 3.3. Toxicological Evaluation Throughout the studies quoted (unless otherwise specified) the purity of the test material used was 99.02%, with an active content of 70.02 to 84.02% w/w, water content of 15% to 29% w/w and total impurities of 0.26 to 0.33% w/w. 3.3.1. Acute toxicity 3.3.1.1. Acute oral toxicity Taken from SCCNFP/08441/04 Guideline: Method: Species/strain: Group size: Test item: Test substance: Batch: Purity: Dose: Vehicle: Route: GLP: / EPA OPP 81-1 Rat, Sprague-Dawley derived, albino 30 (3 groups of 5 males and 5 females each) Benzisothiazolinone Nuosept BIT Technical # 170-138 (PSL Code no E50629-1R (powder) 1,2-Benzisothiazolin-3-one 82.3%; water 17.7% 1000, 2000 and 5000 mg/kg bw/day test substance (823, 1646 and 4115 mg/kg active ingredient) Water; the test substance was applied as 40% w/w suspension in water oral intubation/gavage in compliance Results Based on the findings, the Acute Oral Defined LD50 of Nuosept BIT Technical, Lot #170-138 calculated by Probit Analysis was 1450 milligrams of the test substance per kilogram of bodyweight (when administered as a 40% w/w suspension in distilled water) with 95% Confidence Limits of 2004 mg/kg bw (upper) and 1.049 mg/kg bw (lower). The LD50 for males was 2.100 mg/kg bw with 95% Confidence Limits of 5.029 mg/kg bw (upper) and 877 mg/kg bw (lower). The data does not permit calculation of the LD50 for females by Probit Analysis. Graphically, the LD50 for females was estimated to be 1.050 mg/kg bw. Ref.: 10 9 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 3.3.1.2. Acute dermal toxicity Taken from SCCNFP/08441/04 Guideline: Method: Species/strain: Group size: Test item: Test substance: Batch: Purity: Dose: Vehicle: Route: Exposure period: GLP: / EPA OPP 81-2 rat, Sprague-Dawley derived, albino 10 (5 male/5 female) benzisothiazolinone nuosept BIT Technical # 170-138 (PSL Code no E50629-1R (powder) 1,2-Benzisothiazolin-3-one 82.3%; water 17.7% 5000 mg/kg bw/day (Limit test) (4115 mg/kg active ingredient) water; the test substance was moistened with water for application (1 ml water/1 g test substance) topical application (24 h) 1 x 24 h. observation period 14 d in compliance Results An Acute Dermal Toxicity test was conducted with rats to determine the potential for Nuosept Bit Technical, Lot # 170-138 to produce toxicity after topical application. Based on the results of testing, the single dose Acute Dermal Toxicity LD50 of the test substance is greater than 5000 mg/kg bw when applied as received, moistened with distilled water. 5000 mg of the test substance per kilogram of bodyweight was moistened with distilled water and applied to the skin of ten healthy rats (224-232 g) for 24 hours. The animals were observed for signs of gross toxicity and mortality at least once daily for another 14 days. Bodyweights were recorded just prior to application and again on days 7 and 14 (termination). Necropsies were performed on all animals at terminal sacrifice. All animals survived, gained weight and appeared active and healthy during the study. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. Gross necropsy findings at terminal sacrifice were generally unremarkable. Ref.: 11 3.3.1.3. Acute inhalation toxicity No data submitted 3.3.2 Irritation and corrosivity 3.3.2.1. Skin irritation Taken from SCCNFP/08441/04 (with some modifications) Guideline: Method: Species/strain: Group size: Test substance: Batch: Purity: Dose: Exposure: Exposure time: Readings: GLP: / EPA OPP 81-5 New Zealand albino rabbits 6, 3 males and 3 females Nuosept BIT Technical # 170-138 1,2-Benzisothiazolin-3-one, 82.3%; Water, 17.7% Slurry of 0.5 g in 0.5 ml water Semi occlusive 4 hour(s) 1, 24, 48, 72 hours and 7 days in compliance 10 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Date: 1995 Results The test substance was moistened with water for application (0.5 ml water/0.5 g test substance =41.15% a.i.). One hour after patch removal, well-defined moderate erythema and oedema was noted at all treated sites. This decreased with time. Desquamation occurred at one site and all animals were free of erythema and oedema by day 7. Conclusion Nuosept BIT Technical (Benzisothiazolinone) is a skin irritant. Ref.: 15 Human study 50 healthy human volunteers (20 males and 30 females aged between 19-60 years) were recruited for a randomised double blind open epicutaneous application study to compare the effects of a cream with and without the preservative Microcare® SI. The protocol was approved by an independent ethics committee and the test was performed in compliance with the principles of the Declaration of Helsinki. The test substance was Microcare® SI - an aqueous blend of 2.5% of methylisothiazolinone (MIT) and 2.5% of benzisothiazolinone (BIT). The test cream contained Microcare® SI at a level of 0.3% w/w of which 0.15% w/w (75 ppm) was 1,2-benzisothiazolinone. Analysis of the test products showed that the formulation contained slightly lower concentrations of isothiazolinones than expected, equivalent to an addition rate of 0.2-0.25%, such as would be used in cosmetic applications. Subjects with known sensitivity to isothiazolinones were excluded. 47 subjects completed the study as planned, and diary cards and product weights were available. Three subjects withdraw for reasons not related to the study. The test subjects applied twice daily for 4 weeks 1.5 ml of test cream and vehicle to the inner aspects of both forearms in a randomised and blinded fashion. The test sites were assessed after 2 and 4 weeks and the skin reactions scored according to a 5 point ranking scale. Results 7 subjects reported redness or itching, tingling or stinging sensation upon application of the test cream. The reaction was reported to disappear after the product had been ‘absorbed’ into the skin. Determining causation demonstrated that all 7 subjects experienced sensations following application of the base cream whereas only 5 experienced sensations following the test cream. Conclusion A skin cream preserved with a mixture of MIT/BIT 1:1 at a concentration of 75 ppm of each active was tolerated as well as the vehicle cream under the conditions of the test. Ref.: 22 3.3.2.2. Mucous membrane irritation Taken from SCCNFP/08441/04 Guideline: Method: Species/strain: Group size: Test substance: Batch: Purity: / EPA OPP 81-4 New Zealand albino rabbits 9 (4 males and 5 females) Nuosept BIT Technical # 170-138 1,2-Benzisothiazolin-3-one, 82.3%; Water, 17.7% 11 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Dose: Exposure time: GLP: Date: 0.1 g of the undiluted test substance was instilled into the right eye. The treated eyes of 3 rabbits were rinsed 20-30 seconds after instillation; the eyes of the remaining 6 animals were not rinsed. 48 hours in compliance 1995 Results From 1 to 48 hours, all treated eyes exhibited severe to maximal irritation including corneal opacity, iritis and conjunctivitis. Overall the severity of irritation increased with time. Due to the irreversible nature of the irritation the test was terminated after 48 hours. Conclusion The test substance was severely irritating to the rabbit eye. Ref.: 17 An assessment of the eye irritancy potential of 1,2-benzisothiazolin-3-one using the Bovine Corneal Opacity and Permeability assay in vitro Guideline: Method: Species: Number corneas: Test substance: Batch: Purity: Dose: Exposure time: GLP: Date: / INVITTOX Protocol 124 Bovine 45 1,2-Benzisothiazolin-3-one LHW 1355 >99% 75, 750 and 7500 ppm aqueous solution of BIT prepared as its sodium salt 10 minutes in compliance 2003 Bovine eyes mounted on holders and incubated with Minimal Essential Medium (MEM) were exposed to test article or positive or negative control. After 10 minutes exposure the corneas were rinsed and again incubated with media. Corneal opacity was measured with an opacimeter and corneal permeability was determined using sodium fluorescein and measured spectrophotometrically. The corneal opacity and permeability were combined to give an in-vitro score Results The mean in vitro score for BIT at 7500 ppm was 3.012, at 750 ppm 0.666 and at 75 ppm 0.207, compared with 0.490, 0.416 and 0.449 for saline (negative control) and 50.73, 50.23 and 50.25 for ethanol (positive control), respectively. Conclusion Benzisothiazolinone was considered to be non-irritant to the eye at all tested concentrations in the BCOP assay under the conditions of the test. Ref.: 18 Comment The method used was not a validated in vitro method in 2003. Meanwhile, it is an OECD test guideline (number) which can detect strong irritants. 12 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ An assessment of the cytotoxicity of 1,2 benzisothiazolin-3-one by in vitro Neutral Red Uptake Assay using BalbC 3T3 & SIRC mammalian cell lines Guideline: Species: Test substance: Batch: Purity: Dose: Exposure time: GLP: Date: / BALB/c 3T3 Mouse fibroblast cell line SIRC Rabbit corneal cell line 1,2-Benzisothiazolin-3-one LHW 1355 > 99% 0-100 ppm range finding; 0-10 ppm testing 24 hours in compliance 2002 Results The effects of the test substance on cell viability of the two different cell lines was measured by the neutral red uptake. A best-fit dose-response curve for each set of experiments was calculated from the data using non-linear regression and the respective EC50 value was calculated. The EC50 for 3T3 cells was 3.1414 ppm and that for SIRC cells was 3.6666. These results may be compared with results previously obtained for other preservative preparations, as illustrated in the following table demonstrating that BIT is less cytotoxic than CIT/MIT, yet more cytotoxic than other commonly used preservatives. Cytotoxicity values for cosmetic preservatives Cosmetic Preservative Chloromethylisothiazolinone/methylisothiazolinone (3:1) Methylisothiazolinone Methyldibromoglutaronitrile 20% (in phenoxyethanol) Methyl/ethyl/propyl/butyl/isobutyl parabens in phenoxyethanol EC50 3T3 1.19 5.76 28.4 439.5 EC50 SIRC 1.89 5.98 29.36 489.0 Ref.: 19 Comment The SCCS considers the above cytotoxicity data of little value in its overall evaluation of the safety of benzisothiazolinone. 3.3.3. Skin sensitisation Taken from SCCNFP/08441/04 Guinea Pig Maximization Test (Magnusson and Kligman) Guideline: Species/ strain: Size: Test substance: Batch: Purity: Dosage: Vehicle: GLP: Date: OECD 406 Albino Dunkin Hartley guinea pigs 38 (20 test, 10 control, 8 range-finding) 1,2-benzisothiazolin-3-one 79.8%, water 19.2% 386-3 79.8% 1,2-benzisothiazolin-3-one (BIT), water, 19.2% Diamide content 0.28%, PCP < 1 ppm. 1st induction 0.1% w/v intracutaneous 2nd induction 20% w/v occlusive epicutaneous 3rd challenge 10% w/v occlusive epicutaneous corn seed oil (1st and 2nd concentration), FCA/water (1st concentration), Ethanol (3rd concentration) in compliance 1996 13 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Results Results from 2 animals in range-finding studies indicated that 0.1% w/v in cottonseed oil should be used for intradermal induction. In topical range-finding studies in 4 animals, it was indicated that 20% in cottonseed oil was minimally irritant and was suitable for topical induction. In further topical range-finding studies in 2 animals it was found that 10% in ethanol was suitable for challenge. Following challenge, 9 out of 20 animals in the test group reacted positively to 10% w/v test article in ethanol at 24 or 48-hour examinations, giving a response incidence of 45%. Conclusion BIT is a moderate contact sensitizer. Ref.: 20 Buehler Method Guideline: Method: Species/strain: Size: Test substance: Batch: Purity: Dosage: Vehicle: GLP: Date: / EPA OPP 81-6 Hartley albino guinea pigs 8 for range finding, 30 for test protocol Nuosept Bit technical (82.3% 1,2-Benzisothiazolin-3-one, water 17.7%) #170-138 1,2-Benzisothiazolin-3-one (BIT) 82.3% a.i. Induction: Weekly application of 0.3 g of test substance 95% w/w in corn seed oil for 3 consecutive weeks. Challenge: 14 days after last induction with same dose as induction to naive site Corn seed oil in compliance 1995 Results It was found that a 6 hour exposure under 25 mm Hilltop chambers to 95% w/w (78.19% active) BIT powder in corn oil was suitable for the test group. 0.04% DNCB (dichloronitrobenzene) in acetone was used as the positive control. No reaction was seen at any test or naive control site following challenge. 7/10 positive control animals exhibited signs of reaction to challenge at 24 hours. This reaction persisted in 5 animals at 48 hours. Conclusion Nuosept BIT Technical was not a sensitizer in this test. Ref.: 21 Human study 15 healthy human volunteer patients (2 males and 13 females aged between 25-66 years) were recruited for a randomised double blind open epicutaneous application study to compare the effects of a cream with and without the preservative Microcare® SI. The volunteer subjects were previously diagnosed as being sensitized to chloromethylisothiazolinone. The test cream Doublebase™ contained Microcare® SI at a level of 0.3% w/w (150ppm) of which 0.15% w/w (75ppm) was 1,2-benzisothiazolinone. The test subjects were instructed to apply twice daily for 4 weeks 1-1.5 ml of test cream and vehicle to the inner aspects of both forearms in a randomised and blinded fashion. They were asked to complete a diary to record usage of the products, which were weighed at the end of study. 14 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Results 10 subjects completed the study as planned, and diary cards and product weights were available. Three subjects were withdrawn from the study due to adverse reactions, 2 subjects after less than 7 days and 1 after 21 days. They all noticed flare of eczema on their forearms. Two subjects were lost to follow up. After four weeks application, the frequency of each assessment grade is summarised as follows. Assessment grading Code Withdrawn No Assessment No Visible Redness Slight Redness Distinct Redness Total Product Product Code 1 3 2 9 0 1 15 2 3 2 10 0 0 15 There were three cases of flare of eczema related to the study preparations. Two were associated with the application of product 1, and one with product 2. Ref.: 23 Comments The study included a low number of test subjects; there was no detailed description of their existing chloromethylisothiazolinone (CMI) allergy, lack of assessment of skin reactions under the 4 week study period, and a significant variation in product usage, from around 35 grams to 110 grams per test period. Limited conclusions can be drawn. The test products seem to elicit dermatitis in some test subjects. Local Lymph Node Assay and Human Repeat Insult Patch Test The relative sensitising potencies and potential for cross sensitisation/reaction of chloromethylisothiazolinone (CMI or CIT/MIT), methyl trimethylene isothiazolinone (MTI) and benzisothiazolinone (BIT) were considered from newly generated and historical data. However, although CMI was specifically mentioned, it was not stated whether the mixture of chloromethylisothiazolinone with methylisothiazolinone was actually used. Original experimental data were not provided in this review. Using the LLNA, the EC3 for Benzisothiazolinone was established at 10.4%, that for MTI at 2% and CIT/MIT at 0.01%. Using the HRIPT resulted in no reactions to BIT at 360 ppm and 9% of volunteers reacting at 725 ppm. There were also no reactions to CMIT/MIT at 10 ppm and 4.4% reacted at 20 ppm. For MTI there were no reactions at 100 ppm but 16% reacted at 300 ppm. Data are summarized: Preservative BIT BIT MTI MTI CIT/MIT CIT/MIT Test concentration (ppm) 725 360 300 100 20 10 Proportion of human panel sensitised (%) 5/58 (9%) 0/54 (0%) 3/19 (16%) 0/211(0%) 2/45 (4.4%) 0/175(0%) LLNA EC3 10.4% 2.0% 0.007-0.01% Ref.: 24 Comment The HRIPT is not considered ethical. 15 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Local Lymph Node Assay An EC3 of 2.3% for 1,2-benzisothiazolin-3-one (CAS 2634-33-5) and 1.9% for 2-methyl2H-isothiazol-3-one (CAS 2682-20-4) is tabulated in a review but no experimental details are provided. (Add. ref.: 49) Overall Comment Sensitisation Benzisothiazolinone is a contact allergen with the guinea pig maximization (Magnusson Kligman) test indicating BIT as a moderate sensitiser, as does the LLNA with an EC3 of 2.3% for BIT. Since the human in-use study provided by the applicant included a low number of subjects, only limited conclusions could be drawn. Therefore, the SCCS conducted a literature search on the sensitising potential of benzisothiazolinone in humans. From the dermatological literature case reports describe allergic contact dermatitis to benzisothiazolinone. It is a well-documented contact allergen. However, its potency is lower than other marketed cosmetic preservatives, and the irritancy profile makes it a difficult contact allergen to test with. (Add. ref.: 37, 38, 39, 40) Benzisothiazolinone is widely used in industry as a preservative in water-based solutions such as pastes, paints and cutting oils. Occupational dermatitis has been reported mainly due to cutting fluids, paint manufacture, pottery mould-makers, acrylic emulsions manufacture, printers, paper makers etc. which contain benzisothiazolinone. (Add. ref. 42, 44, 45, 46, 47, 48) Moreover, benzisothiazolinone is used as a slimicide in the manufacture of disposable powder-free polyvinyl chloride (PVC) gloves. Of 31 glove brands studied 9 (30%) contained 3–26 ppm benzisothiazolinone. Individuals wearing such gloves developed allergic contact dermatitis. A concentration of 20 ppm benzisothiazolinone in a glove seems to be enough for sensitization. (Add. ref. 41) 3.3.4. Dermal / percutaneous absorption New study Guideline: Species/strain: Group size: Method Membrane integrity Test substance: Batch: Purity: Radiochemical Vehicle: Test item: Dose volume: Receptor Solubility Method of Analysis: GLP: Study period: OECD 428 human skin (5 abdominal, 1 breast); 400 μm dermatomed skin 10 membranes flow through diffusion cell Tritiated water benzisothiazolinone 12409EE 97% 14 C benzisothiazolinone; 99.9% water 0.01% w/v benzisothiazolinone aqueous 20 μl/cm2 Tissue culture medium containing 6% PEG, 0.01% sodium azide, 1% glucose and streptomycin (in water) 1 mg/ml liquid scintillation in compliance 2008 16 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ The absorption of radiolabelled 0.01% w/v benzisothiazolinone in aqueous solution was determined from the use of 10 chambers with human dermatomed skin. 17 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Conclusion Following topical application of [14C]-Benzisothiazolinone in water (0.01%, w/v) to human skin, the absorbed dose of [14C]-Benzisothiazolinone was 25.63% (0.54 μg equiv./cm2). The dermal delivery of [14C]-Benzisothiazolinone was 49.83% (1.04 μg equiv./cm2). The total dislodgeable dose was 42.05% of the applied dose. Ref.: 36 Comment This was a properly performed study. Therefore, the mean + 1SD may be used for calculating the MOS. This is 61.9% of the applied dose (1.29 μg/cm2) when 0.01% benzisothiazolinone aqueous was applied. The dermal absorption studies had not been performed with representative cosmetic formulations. 3.3.5. Repeated dose toxicity 3.3.5.1. Repeated Dose (28 days) oral toxicity Taken from SCCNFP/08441/04 (with modifications) Guideline: Species/strain: Group size: Test item: Test substance: Purity: Batch: Dose levels: Route: Exposure period: GLP: Date: OECD 407 Rat, Wistar Hsd Cpb:WU 12 (6 male/6 female) Benzisothiazolinone, Code 072/1-PBP Promex BIT (paste) 1,2-Benzisothiazolin-3-one, 84.29%; water, 15%, purity of active ingredient on dry weight basis, 99.02% 2001 014//sample no. KP 070601//cb 181100 0, 15, 45 and 135 mg/kg bw/day (12.63, 37.89 and 113.67 mg/kg a.i.), suspended in 0.5% CMC daily oral intubation/gavage 28 days in compliance 2001 Results 18 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Oral administration of 1,2-Benzisothiazolin-3-one, by gavage in Wistar rats at the dose of 15 mg/kg bw/day (12.63 mg a.i./kg bw/day) had no adverse effect on general health, neurological effects, growth, food consumption, haematological and clinical chemistry parameters, organ weights and its ratios, gross and histopathological changes. Treatment related signs of slight salivation were observed in all the males in the main group at 135 mg/kg bw/day and its recovery group from treatment day 17 and in two females from the test group and two in the recovery group from treatment day 20 till the end of the treatment period. During the recovery period, treatment related signs of salivation were not observed in the 135 mg/kg bw/day group indicating the reversibility of the effect. Body weight was unaffected in the 15 and 45 mg/kg bw/day groups. At 135 mg/kg bw/day there was a significant decrease in body weight and cumulative net weight gain in the male group throughout the treatment period with the exception of the first week where it was not statistically significant. Weekly body weights were significantly lower throughout the treatment and recovery period in the high dose (135 mg/kg bw/day) for males and on weeks 4, 5, and 6 for females. Cumulative net weight gains were also significantly lower throughout the treatment and recovery period in males and females with the exception of the first week where it was not statistically significant. Increased incidences of histopathological lesions in the non-glandular stomach (hyperkeratosis, epithelial hyperplasia, ulceration) were observed in mid- (45 mg/kg bw/day) and high-dose (135 mg/kg bw/day) males and females. The severity of the lesions was reduced in the high-dose recovery group. The NOAEL in this study was 15 mg/kg bw/day (12.63 mg a.i./kg bw/day). Ref.: 12 Comment The SCCS noted that the NOAEL of 15 mg/kg bw/day (12.63 mg a.i./kg bw/day) was based on the histopathological lesions observed in the non-glandular stomach, which are most likely due to the irritant property of the test substance. 3.3.5.2. Sub-chronic (90 days) toxicity (oral, dermal) Taken from SCCNFP/08441/04 Guideline: Species/strain: Group size: Test item: Batch: Purity: Dose levels: Vehicle: Route: Exposure period: GLP: Study period: OECD 408 Rat, Wistar Total 20; 10 male/10 female Benzisothiazolinone (Promex-BIT) G00Z-0600-1907-13//072/1.PBP//2001 014//KP 070601 1,2-Benzisothiazolin-3-one, 84.29%; water 15%; purity of active ingredient on dry weight base 99.1% 10, 30 and 75 mg/kg bw/day (8.42, 25.26 and 63.15 mg/kg bw/day a.i.) 0.5% carboxymethylcellulose (CMC) daily oral intubation/gavage 90 days in compliance 2001-2002 Results The oral administration of 1,2-Benzisothiazolin-3-one by gavage in Wistar rats at the dose of 10 mg/kg bw/day (8.42 mg a.i./kg bw/day) had no adverse effect on general health, neurological effects, growth, food consumption, haematological and clinical chemistry parameters, sperm evaluation, organ weights and its ratios and gross and histopathological changes. 19 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ At 30 mg/kg bw/day (25.26 mg a.i./kg bw/day) there were no treatment related clinical signs or neurological effects and no adverse effects on growth, haematological and clinical chemistry parameters, sperm evaluation, organ weights and its ratios. Food consumption was lower in females in weeks 2 and 4. There were some changes primarily in the nonglandular stomach region both macroscopically and histologically which were considered treatment related and were reversible. These effects may have been due to the irritant nature to the test substance. Increased incidences of histopathological lesions in the nonglandular stomach (hyperkeratosis, epithelial hyperplasia, ulceration) were observed in males and females. At 75 mg/kg bw/day (63.15 mg a.i./kg bw/day) there were no treatment related neurological effects and no adverse effects on haematological and clinical chemistry parameters, sperm evaluation, organ weights and its ratios. Treatment related signs of slight salivation were observed in four males and three females in the main group and in three males and five females in the recovery group during the treatment period. During the recovery period, treatment related signs of salivation were not observed indicating the reversibility of the effect. Weekly body weights and cumulative net weight gains were significantly lower in males throughout the treatment and recovery period except for the body weight at week 3 which were lower but not statistically significant. The body weight gains were significantly higher in males and in females (weeks 15, 16 and 17) during the recovery period. There was a significant reduction in food consumption in males (weeks 1, 2, 7, 8, 12 and 13) and in females (weeks 1, 2 and 4), which returned to control levels in the recovery period. Increased incidences of histopathological lesions in the non-glandular stomach (hyperkeratosis, epithelial hyperplasia, ulceration, keratin cysts) were observed in males and females. The severity of the lesions was reduced in the recovery group. The NOAEL in this study was 10 mg/kg bw/day (8.42 mg a.i./kg bw/day). Ref.: 13 Comment The SCCS noted that the NOAEL of 10 mg/kg bw/day (8.42 mg a.i./kg bw/day) was based on the histopathological lesions observed in the non-glandular stomach, which are most likely due to the irritant property of the test substance. Therefore, the NOAEL is 25.26 mg a.i./kg bw/day based on systemic effects. 3.3.5.3. Chronic (> 12 months) toxicity No data submitted 3.3.6. Mutagenicity / Genotoxicity 3.3.6.1 Mutagenicity / Genotoxicity in vitro Taken from SCCNFP/08441/04 Bacterial Reverse Mutation Assay Guideline: Species/strain: Test substance: Batch: Lot number: Purity: Concentrations: OECD 471 (1997) Salmonella typhimurium TA98, TA1537, TA100, TA1535 Escherichia coli WP2uvrA pkM 101 Promex BIT 1,2–Benzisothiazolin-3-one 2001 014 KP 070601 99.02% 0, 20, 35, 60, 100 and 175 μg/plate (1st experiment) 0, 30, 50, 75, 120 and 180 μg/plate (2nd experiment) 20 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Replicate: Positive controls: Metabolic activation: GLP: Date: 3 plates/concentration according to guideline Aroclor induced rat liver homogenate in compliance 2001 Results Toxicity: in a preliminary study with a series of concentrations up to 5000 μg/plate, there was a decrease in the mean number of revertants from the concentrations up to 160 μg/plate. Mutagenicity: only the lowest doses could be evaluated in comparison with the untreated plates (10-20 μg/plate). The study cannot be used for the evaluation due to the high toxicity of the test item towards the bacterial cells. Ref.: 25 In vitro Mammalian Cell Gene Mutation Test Guideline: Species/strain: Test substance: Batch: Lot number: Purity: Concentrations: Treatment time: Replicate: Positive controls: Metabolic activation: GLP: Date: OECD 476 (1997) CHO-K1 (Chinese hamster ovary cells) HPRT locus Promex BIT; 1,2-Benzisothiazolin-3-one 2001 014 KP 070601 99.02% 0, 0.65, 1.30, 2.60, 5.20 μg/ml 5 hours, with and without metabolic activation 2 experiments in the same conditions. B(a)P; EMS Aroclor 1254 induced rat liver homogenate. in compliance 2002 Results Toxicity: in the presence of metabolic activation a toxic effect produced by the test item between 4 and 6 μg/ml was observed; in the absence of metabolic activation a toxic effect produced by the test item was observed between 2 and 4 μg/ml. The toxic doses reduced the survival to less than 50% of the untreated cells. Mutagenicity: there was no increase of mutants in the treatment with the test substance, in the presence and in the absence of metabolic activation after 5 hours of treatment. In the absence of metabolic activation, a treatment of 20 hours was not performed as suggested by the guideline. The study indicates that the test item is not mutagenic in the condition of the test. Ref. 26 In vitro Mammalian Chromosome Aberration test Guideline: Species/strain: Test substance: Batch: Lot number: Purity: Concentrations: Replicate: OECD 473 (1997) CHO-K1 cell line (Chinese hamster ovary cells) Promex BIT; 1,2–Benzisothiazolin-3-one 2001 014 KP 070601 99.02% 0, 1.6, 3.2, 6.4 μg/ml in the presence of S9-mix 0, 1.25, 2.50, 5.0 μg/ml in the absence of S9-mix 2 experiments (200 metaphases analysed) 21 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Treatment time: Positive controls: Metabolic activation: GLP: Date: 1st experiment: 3 hours 2nd experiment: 3 hours, in the presence of S9-mix; 19 hours, in the absence of S9-mix CPA (55 μg/ml); EMS (600 μg/ml) Aroclor 1254 induced rat live homogenate in compliance 2001 Results Toxicity: 2 preliminary experiments showed that the test item was toxic at concentrations between 75 and 5000 μg/ml and between 14 and 58.94 μg/ml. Clastogenicity: the test item induced chromosome aberrations at the maximum tested dose in the presence of a metabolic activation, and at all concentrations, in the absence of a metabolic activation system. The test item is clastogenic in CHO mammalian cells. Ref.: 27 3.3.6.2 Mutagenicity / Genotoxicity in vivo Taken from SCCNFP/08441/04 Mammalian Erythrocyte Micronucleus Test Guideline: Species/strain: Test substance: Batch: Lot number: Purity: Doses: Treatment: Positive control: GLP: Date: OECD 474 (1997) Swiss albino mice-HsdOla: MF1 strain Promex BIT; 1,2–Benzisothiazolin-3-one 2001 014 KP 070601 99.02% 63.15; 126.3; 210.5 mg/kg a.i. oral (gavage) twice, at 24 hours of interval. The animals were sacrificed 24 hours after the second treatment. CPA, 40 mg/kg bw, oral treatment in compliance 2001 Results Toxicity: in a preliminary test, a dose of 250 mg/kg bw was found not toxic (no clinical signs), whereas 450 and 900 mg/kg bw were toxic. Clastogenicity: in all treated mice, there was a reduction of the ratio PCE/NCE, thus indicating that the test item has reached the target cells. The positive control, CPA, induced a number of MN significantly higher than the untreated animals. The test item did not induce a number of MN higher than the untreated animals. The test item is not clastogenic in mice, treated in vivo. Ref.: 28 Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo Guideline: Species/strain: Test substance: Batch: Purity: Doses: Treatment times: OECD 486 (1997) Wistar Hanlbm: WIST (SPF) rats Promex BIT 2001014 99.02% 0 (corn oil), 375, 750 mg a.i./kg bw 2 hours, 16 hours, orally, once 22 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Positive controls: GLP: Date: N,N’-dimethylhydrazine dihidrochloride (DMH): 40 mg/kg bw; 2 hours 2acetylaminofluorene (2-AAF): 100 mg/kg bw; 16 hours. in compliance 2002 Results Toxicity: in preliminary experiments, doses of 1200 and 1000 mg a.i./kg bw were found toxic to the animals. DNA repair: autoradiography was done on at least three cultures of hepatocytes per animals. There was no indication of induction of UDS by the test item. The two positive controls induced a significant increase of UDS. The test item does not induce UDS in rat hepatocytes in in vivo treatment. Ref.: 29 3.3.7. Carcinogenicity No data submitted 3.3.8. Reproductive toxicity 3.3.8.1. Two generation reproduction toxicity study Guideline: Species/strain: Group size: Test substance: Batch: Purity: Dose levels: Route: Administration: Control: GLP statement: Study period: EPA Health Effects Test Guidelines OPPTS 870.3800, of aug 1998 Crl:W1 (Glx/BRL/Han)BR 24 males and 24 females (P generation) Proxel Press Paste (1,2-Benzisothiazolin-3-one) No. 103 and No. 344 Purity of active ingredient: 92.7% +/- 1.1% P generation: 250, 500 and 1000 ppm F1 generation: 250, 500 1000 ppm oral dietary control diet only Yes 2000-2001 Study design Groups of 24 male and 24 female rats were given Proxel Press Paste by admixture with the diet, at dose levels of 250, 500 and 1000 ppm. A similar group received the control diet only. The animals received the test diet for 10 weeks before being paired for up to 2 weeks. Dosing continued during this pairing period, and throughout the resulting pregnancies. The P generation females were allowed to litter and rear their offspring (F1a) until weaning. Administration of the test article continued throughout the weaning of the F1 offspring up until necropsy. 24 animals of each sex were randomly selected from each group to form the filial (F1) generation. Direct treatment of the F1 generation continued during their maturation period (10 weeks), the mating period (up to two weeks) and throughout the resulting pregnancies and weaning of the F2 offspring until necropsy. All F1 females were allowed to litter and rear their F2a offspring to weaning. Result Analysis of samples from the diets prepared for administration in weeks 1, 17 and 19 of the study showed that the achieved concentrations were within the target range (all values within 89 to 110% of nominal). Analysis of samples from the diets prepared for 23 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ administration in weeks 34, 36, 37 and 38 of the study showed that the achieved concentrations were below the target range. P Generation: Clinical observations, body weights and food intakes were unaffected by treatment. Mating data, duration of gestation, number of implantations, numbers of pups born and pup survival were similar in all groups. Mean pup weight of the high dose pups was slightly lower than the control over the first week post-partum. But, over the whole lactation period, mean pup weight was similar in all groups. There were no adverse effects of treatment on seminology data. In the high dose group, mean liver weight of the males was slightly higher, and mean testes weight slightly lower than control. Minor limiting ridge hyperplasia in the stomach was noted in some intermediate and many high dose animals. Squamous cell hyperplasia and forestomach gastritis was also seen in a few animals. F1 Generation: Males in the high dose group gained slightly less weight than the controls during the study and the high dose females gained slightly less weight during the prepairing period only. Clinical observations and food intakes were unaffected by treatment. Physical development of the F1 generation, mating data, duration of gestation and F2a pup sex ratio were unaffected by treatment. Pup survival to day 4 post-partum and mean pup weight gain were slightly lower in the high dose group compared to controls. Seminology investigations, organ weights and ovarian follicle counts were unaffected by treatment. In the intermediate and high dose groups, limiting ridge hyperplasia in the stomach was noted. This was most prominent in the high dose females where there was also squamous cell hyperplasia, fore stomach gastritis, hyperkeratosis and erosion/ulcer. Conclusion Dietary administration of 1000 ppm PROXEL Press Paste to rats for two generations produced slight adult toxicity, in the F1 generation in terms of lower body weight gain, and in both generations limiting ridge hyperplasia of the stomach together with incidences of squamous cell hyperplasia, forestomach gastritis, hyperkeratosis and erosion/ulcer. At this concentration, the growth of the offspring was slightly impaired and in the F2a offspring, there was a slight reduction in pup survival. At 500 ppm, there were incidences of limiting ridge hyperplasia in the stomach only. There were no adverse effects of treatment at 250 ppm, equivalent to an approximate overall mean intake of 24 mg/kg bw/day. Note: The dose levels refer to the concentration of the active ingredient (BIT) and a correction factor of 1.074 was made for the stated purity of the PROXEL. Ref.: 14 24 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ Comments No significant effects were reported for the reproductive parameters at any dose level and only slight effects were noted in the offspring at the highest dose level (slightly lower pup survival to day 4 post-partum and slightly lower mean pup weight gain). Based on this study, a NOAEL of approximately 50 mg a.i./kg bw/day is used as a conservative estimate for the MOS calculation. This NOAEL is lower than the lowest LOAEL for systemic effects (63 mg a.i./kg bw/day) in the 90-day study. 3.3.8.2. Teratogenicity No evidence for teratogenicity in the two-generation oral reprotoxicity study 3.3.9. Ref.: 14 Toxicokinetics No data provided. 3.3.10. Photo-induced toxicity 3.3.10.1. Phototoxicity / photoirritation and photosensitisation Guideline: Test: Test substance: Batch: Purity: UV-A Irradiation: Concentrations: Replicate: Positive control: GLP: Date: OECD Test guideline 432 Neutral Red uptake phototoxicity test with Balb/c 3T3 cells 1,2–Benzisothiazolin-3-one LMS 414, CH-0405-ST-7 > 99% SOL-500 lamp fitted with a H1-filter mounted at a distance of 60 cm to achieve an UV-irradiance of 5 J/cm2 0, eight concentrations between 0.2 to 50 µg/mL in the presence and absence of UV-A exposure 3 main experiments Chlorpromazine in compliance 2004 Results The 3T3 NRU phototoxicity assay was performed on four occasions for BIT, once as a range finding experiment and three times as main experiment. The PIF values calculated from the results for BIT range between 1.197 and 2.166 with a mean value of 1.54. The positive control chlorpromazine yielded PIF values of about 100. Conclusion 1,2 Benzisothiazol-3(2H)-one is predicted to be non-phototoxic by the 3T3 NRU PT assay OECD Test Guideline 432 Ref.: 16 3.3.10.2. Phototoxicity / photomutagenicity / photoclastogenicity No data submitted 3.3.11. Human data See section 3.3.3. Sensitisation 25 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 3.3.12. Special investigations No data submitted 3.3.13. Safety evaluation (including calculation of the MoS) CALCULATION OF THE MARGIN OF SAFETY Benzisothiazolinone Daily exposure to all cosmetic products (excl. sunscreens) = Concentration Benzisothiazolinone (BIT) = Daily exposure BIT = Dermal absorption = Typical body weight of human = Systemic exposure dose = No Observed Adverse Effect Level = (2-generation-study, oral, rat) NOAEL corrected for 50% oral bioavailability = 17.4 g/d 0.01% 1.74 mg 61.9% 60 kg 0.018 mg/kg bw/d 50 mg/kg bw/d Margin of Safety 1392 3.3.14. NOAEL/SED = 25 mg/kg bw/d Discussion Physico-chemical properties Benzisothiazolinone is an off-white to yellowish solid that is soluble in water (1.1 g/L at 20°C); its log Pow shows a significant dependence on the pH. No data on stability are provided. Irritation, sensitisation According to a study conducted in rabbits benzisothiazolinone (BIT) can be classified as a moderate irritant to skin. A study in rabbits classified the compound as a severe eye irritant. A guinea pig maximization test classified BIT as a moderate contact sensitizer whilst the Buehler test classifies BIT as non-sensitising. Literature data for the local lymph node assay support a classification of BIT as a moderate dermal sensitizer (EC3 2.3%). From a four week in-use study with humans only limited conclusions can be drawn due to the low number of subjects, no detailed description of pre-existing chloromethylisothiazolinone allergy and a significant variation in product use. Several case reports in the dermatological literature describe allergic contact dermatitis to benzisothiazolinone, and some individuals wearing disposable powder-free polyvinyl chloride gloves containing 3 - 36 ppm benzisothiazolinone have developed allergic contact dermatitis (Aalto-Korte et al. 2007). According to the latter study, a concentration of 20 ppm benzisothiazolinone in a glove seems to be enough for sensitization. Moreover, in the context of occupational uses, benzisothiazolinone (BIT) is a well-documented contact allergen. As has been seen with MCI/MI and now with MI itself, these isothiazolinones are important contact allergens for the consumer in Europe. Within the mixture, MCI is known to be the more potent allergen (EC3 0.009%). MI is less potent (EC3 1.9%) and is now permitted at up to 100 ppm in leave on and rinse off cosmetic products; contact allergy to MI itself is 26 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ now a considerable problem in Europe and this is of concern (Add. ref: 50-61). If BIT is to be used as a preservative in cosmetic products, it is essential that the level be sufficiently low to prevent a repeat of history. It is recommended that the incidence of contact allergy to BIT and other isothiazolinones be monitored at regular intervals (e.g. annually), by reference to dermatology clinic data in Europe. Necessary early interventions can then be introduced to reduce exposures and hence contact allergy and allergic contact dermatitis as required. Dermal absorption An in vitro study with human skin has been provided. The mean value + 1SD can be used for calculating the MOS. This was 61.9% of the applied dose (1.29 μg/cm2) when 0.01% benzisothiazolinone aqueous was applied. The dermal absorption studies had not been performed with representative cosmetic formulations. General toxicity The acute toxicity of benzisothiazolinone upon oral or dermal administration to rats is low (LD50 of 1193 and 4115 mg/kg bw, respectively). The NOAEL derived from a subacute 28 day oral (gavage) toxicity study in rats was 12.63 mg a.i./kg bw/day. Subchronic toxicity was evaluated in a 90 day oral (gavage) study (according to OECD guideline 408) and provided a NOAEL of 8.42 mg/kg bw/day for the active ingredient. The NOAELs in the 28day and 90-day studies were based on histopathological lesions observed in the nonglandular stomach, which are most likely due to the irritant property of the test substance and are therefore, not relevant for the safety assessment of benzisothiazolinone as a cosmetic ingredient. Reproductive Toxicity In a two generation study in rats with dietary administration benzisothiazolinone produced at 1000 ppm slight adult toxicity in the F1 generation in terms of lower body weight gain, and in both generations limiting ridge hyperplasia of the stomach together with incidences of squamous cell hyperplasia, forestomach gastritis, keratosis and erosion/ulcer. At this concentration, the growth of the offspring was slightly impaired and in the F2a offspring, there was a slight reduction in pup survival. At 500 ppm, there were incidences of limiting ridge hyperplasia in the stomach only. There were no adverse effects of treatment at 250 ppm, equivalent to an approximate overall mean intake of 24 mg/kg bw/day (active ingredient). The NOAEL was based on histopathological lesions observed in the nonglandular stomach, which are most likely due to the irritant property of the test substance and therefore, not relevant for the safety assessment of benzisothiazolinone as a cosmetic ingredient. Therefore, the NOAEL of 50 mg a.i./kg bw/day for the systemic effects of benzisothiazolinone will be used for the safety assessment. Mutagenicity Benzisothiazolinone has been tested for the induction of gene mutation in bacterial and mammalian cells treated in vitro, for clastogenicity on mammalian cells treated in vitro, for the induction of micronuclei in mice and for the induction of UDS in rats treated in vivo. The study on the induction of gene mutations on bacterial cells is inadequate due to the toxicity of the test item. The compound has been found to be clastogenic in mammalian cells treated in vitro. The compound has been found non mutagenic in vitro, non clastogenic and DNA damaging in vivo. Carcinogenicity No data provided 27 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 4. CONCLUSION 1. Does SCCS consider benzisothiazolinone safe when used as a preservative up to a maximum authorised concentration of 0.01% in cosmetic products, based on the provided data? The SCCS considers benzisothiazolinone safe for use as a preservative in cosmetics products up to 0.01% with respect to systemic toxicity. However, its sensitising potential is of concern. 2. And/or does the SCCS have any scientific concern with regard to the use of benzisothiazolinone in cosmetic products? Sensitisation from related isothiazolinones is an important problem in consumers. This has occurred because there has been consumer exposure before safe levels of exposure relevant to sensitisation have been established. Benzisothiazolinone is a skin sensitiser in animal models with potency similar to methylisothiazolinone. Methylisothiazolinone, at 100 ppm (0.01%) in cosmetic products is causing contact allergy and allergic contact dermatitis in the consumer. Benzisothiazolinone is known to be a sensitiser in man and has induced sensitisation at circa 20 ppm in gloves. There is no information on what may be safe levels of exposure to benzisothiazolinone in cosmetic products from the point of view of sensitisation. Until safe levels of exposure have been established, the use of benzisothiazolinone in cosmetic products as a preservative or for other functions cannot be considered safe in relation to sensitisation. 5. MINORITY OPINION Not applicable 28 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 6. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Material Safety Data Sheet, Acticide BIT, Thor GmbH 22-11-2001 RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702731 "Determination of the Melting Point/Melting Range of ACTICIDE BIT" (Sponsor: THOR Chemie GmbH), 22-10-1998 RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 840980 "Determination of the Boiling Point/Boiling Range of 1,2-Benzisothiazol-3(2H)-one" (Sponsor: Thor GmbH), 2002 Thor Specialities (UK) Limited, Study No: RS/01/025 "Determination of the Density of 1,2-benzisothiazol-3(2H)-one (BIT)" (Sponsor: Thor GmbH), 18-01-2002 RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702696 "Determination of the Vapour Pressure of ACTICIDE BIT" (Sponsor: THOR Chemie GmbH), 19-11-1998 RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702718 "Determination of the Partition Coefficient (n-Octanol/Water) of ACTICIDE BIT" (Sponsor: THOR Chemie GmbH), 22-10-1998 Thor Specialities (UK) Limited, Study No: RS/01/025 "Determination of the Partition Coefficient (noctanol/water) of 1,2-benzisothiazol-3(2H)-one (BIT) at a range of temperatures and pHs" (Sponsor: Thor GmbH), March 2002 RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702431 "Determination of the Water Solubility of ACTICIDE BIT" (Sponsor: THOR Chemie GmbH), 19-11-1998 Thor Specialities (UK) Limited, Study No: RS/01/029 "Determination of the water solubility of 1,2-benzisothiazol-3(2H)-one (BIT) at a range of temperatures and pHs" (Sponsor: Thor GmbH), 2002 psl - product safety labs, Study No. 3859 "Acute Oral Toxicity Defined LD50" (Sponsor: Hüls America Inc., joint ownership: Prom Chem Ltd and Thor GmbH), 24-01-1996 psl - product safety labs, Study No. 3862 "Acute Dermal Toxicity Limit Test" (Sponsor: Hüls America Inc., joint ownership: Prom Chem Ltd and Thor GmbH), 24-01-1996 Toxicology Department Rallis Research Centre, Study No. 3286/01 "Repeated Dose (28-Day) Oral Toxicity Study by Gavage with Promex BIT in Wistar Rats" (Sponsors: Prom Chem Ltd. and Thor GmbH), 27-02-02 Toxicology Department Rallis Research Centre, Study No. 3287/01 "Repeated Dose (90-Day) Oral Toxicity Study by Gavage with Promex BIT in Wistar Rats" (Sponsors: Prom Chem Ltd and Thor GmbH), 2002 Covance Report Number 18D3114-D6154. PROXEL Press Paste (BIT): Two Generation Oral (Dietary Administration) Reproduction Toxicity Study in the Rat. (Sponsor Avecia Limited) September 2003 psl - product safety labs, Study No. 3861 "Primary Skin Irritation" (Sponsor: Hüls America Inc.), joint ownership: Prom Chem Ltd and Thor GmbH), 24-01-1996 Thor Specialities (UK) Limited, Study RS/02/036. “3T3 Neutral Red Uptake Phototoxicity assay “. (Sponsor: Thor Group Management Limited) March 2005. psl - product safety labs, Study No. 3860 "Primary Eye Irritation" (Sponsor: Hüls America Inc., joint ownership: Prom Chem Ltd and Thor GmbH), 24-01-1996 Thor Specialities (UK) Limited, Study RS/02/036. “An assessment of the eye irritancy potential of 1,2-benzisothiazolin-3-one using the In-vitro BCOP assay“. (Sponsor: Thor Group Management Limited) March 2003. Thor Specialities (UK) Limited, Study RS/02/039. “An assessment of the cytotoxicity of 1,2-benzisothiazolin-3-one by the In-vitro Neutral Red Uptake Assay, using BalbC 3T3 and SIRC mammalian cell lines.“ (Sponsor: Thor Group Management Limited) January 2003 Quintiles England limited, Project Code A/K/42557 "Skin Sensitisation Study In The Guinea Pig Magnusson and Kligman Maximisation Test" (Sponsor: H&C Prom Kemi ApS, joint ownership: Prom Chem Ltd and Thor GmbH), 02-1997 29 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. psl - product safety labs, Study No. 3863 "Dermal sensitization test – Buehler Method" (Sponsor: Hüls America Inc., joint ownership: Prom Chem Ltd and Thor GmbH), 2401-1996 Sequani Consumer Limited. Study No TWP0001. “A four week home-use study to evaluate the tolerance to a topical cream containing a new preservative“. (Sponsor Thor Group Mangement Limited). June 2002. Sequani Consumer Limited. Study No TWP0002 June 2002. “ A four week home-use study to evaluate the tolerance to a topical cream containing a new preservative in subjects sensitized to a mixture of isothiazolinones“. Start of experimental work December 2002 D.A.Basketter, R.Rodford, I.Kimber, I.Smith, J.Wahlberg. Skin sensitisation risk assessment: a comparative evaluation of 3 Isothiazolinone biocides. Contact Dermatitis 1999; 40:150-4. Toxicology Department Rallis Research Centre, Study No. 3288/01 "Bacterial Reverse Mutation Test with Promex BIT" (Sponsors: Prom Chem Ltd and Thor GmbH), 01-0302 Toxicology Department Rallis Research Centre, Study No. 3291/01 "In vitro Mammalian Cell Gene Mutation test with Promex BIT" (Sponsors: Prom Chem Ltd and Thor GmbH), 03-02 Toxicology Department Rallis Research Centre, Study No. 3289/01 "In vitro Mammalian Chromosome Aberration test with Promex BIT" (Sponsors: Prom Chem Ltd and Thor GmbH), 03-02 Toxicology Department Rallis Research Centre, Study No. 3290/01 "Mutagenicity Study - Micronucleus Test in Swiss Albino Mice with Promex BIT" (Sponsors: Prom Chem Ltd and Thor GmbH), 2002 RCC Cytotest Cell Research. Study Number: 736501. "Unscheduled DNA synthesis with 1,2-Benzisothiazolin-3-one“ Study Commissioned June 2002 B.R Alexander (2002) An assessment of the comparative sensitisation potential of some common isothiazolinones. Contact Dermatitis 46, 191-196 Steinberg, D.C. (2000) Measuring synergy. Cosmetics & Toiletries 115(11), 59-62. Küll, F.C., Eisman, P.C., Sylwestrowicz, J.D. & Mayer, R.L. (1961) Mixtures of quaternary ammonium compounds and long chain fatty acids as antifungal agents. Applied Microbiology 9, 538-541. Seal K.J., Alexander B.R., (2001) Special Agents. Global Cosmetic Industry, 169 (6), 24-30 Chapman, J.S., Diehl, M.A., Fearnside, K.B. (1998) Preservative tolerance & resistance. International Journal of Cosmetic Science 20, 31-39. Kroes R, Galli C, Munro I, Schilter B, Tran L-A, Walker R, Wurtzen G. (2000) Threshold of Toxicological Concern for Chemicals Substances Present in the Diet: A Practical Tool for Assessing the Need for Toxicity Testing. Food and Chemical Toxicology 38, 2-3, 255-312 Charles River Laboratories Study No. 782039, Report No. 28709, 2008. The In Vitro Percutaneous Absorption of Radiolabelled Benzisothiazolinone Through Human Skin (Sponsor: Thor Group, Ramsgate Road, Margate, Kent, CT9 4JY, UK) Additional References 37. 38. 39. 40. Ayadi M, Martin P. Pulpitis of the fingers from a shoe glue containing 1,2benzisothiazolin-3-one. Contact Dermatitis 1999; 40: 115-116 Fischer T, Bohlin S, Edling C, Rystedt I, Wieslander G. Skin disease and contact sensitivity in house painters using water-based paints, glues and putties. Contact Dermatitis 1995; 32: 39-45 Taran JM, Delaney TA. Allergic contact dermatitis to 1,2-benzisothiazolin-3-one in the carpet industry. Australas J Dermatol 1997; 38: 42-43 Chew AL, Maibach HI. 1,2-Benzisothiazolin-3-one (Proxel): irritant or allergen? A clinical study and literature review. Contact Dermatitis 1997; 36: 131-136 30 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. Aalto-Korte K, Ackermann L, Henriks-Eckerman ML, Vlimaa J, Reinikka-Railo H, Leppnen E, Jolanki R (2007). 1,2-Benzisothiazolin-3-one in disposable polyvinyl chloride gloves for medical use. Contact Dermatitis 57; 365–370. Burden AD, O’Driscoll JB, Page FC, Beck MH (1994) Contact hypersensitivity to a new isothiazolinone. Contact Dermatitis 30; 179-180 Chew AL, Maibach H (1997) 1,2-Benzisothiazolin-3-one (Proxel®): irritant or allergen? A clinical study and literature review. Contact Dermatitis 23; 131-136 Dias M, Lamarao P, Vale T (1992) Occupational contact allergy to 1,2-benzisothiazol-3one in the manufacture of air fresheners. Contact Dermatitis 36; 205-206 Greig DE (1991) Another isothiazolinone source. Contact Dermatitis 25; 201-202 Sanz-Gallen P, Planas J, Martinez P, Gimenez-Arnau JM (1992) Allergic contact dermatitis due to 1,2-benzisothiazolin-3-one in paint manufacture. Contact Dermatitis 27; 271-272. Geier J, Lessmann H, Dickel H, Frosch PJ, Koch P, Becker D, Jappe U, Aberer W, Schnuch A, Uter W (2004). Patch test results with the metalworking fluid series of the German Contact Dermatitis Research Group (DKG). Contact Dermatitis 51; 118–130. Walker SL, Yell JA, Beck MH (2004). Occupational allergic contact dermatitis caused by 1,2-benzisothiazolin-3-one in a varnish maker, followed by sensitization to benzalkonium chloride in Oilatum® Plus bath additive. Contact Dermatitis 50; 104– 105. Roberts DW, Patlewicz G, Kern PS, Gerberick F, Kimber I, Dearman RJ, Ryan CA, Basketter DA, Aptula AO (2007) Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitisation. Chem. Res. Toxicol; 20; 10191030. Cuesta L, Silvestre JF, Toledo F, Ballester I, Betlloch I (2010). Delayed hypersensitivity to methylchloroisothiazolinone/methylisothiazolinone not detected the baseline series of the Spanish group. Contact Dermatitis; 62; 250-251 García-Gavín J, Goossens A (2010). Moist toilet paper: allergy to the nonhalogenated derivative methylisothiazolinone preservative alone. Archives of Dermatology; 146; 1186 Amaro C, Santos R, Cardoso J (2011). Contact allergy to methylisothiazolinone in a deodorant. Contact Dermatitis; 64; 298-299 Lundov MD, Thyssen JP, Zachariae C, Johansen JD (2010). Prevalence and cause of methylisothiazolinone contact allergy. Contact Dermatitis; 63; 164-167 Gardner KH, Davis MD, Richardson DM, Pittelkow MR (2010). The hazards of moist toilet paper: allergy to the preservative methylchloroisothiazolinone/methylisothiazolinone. Archives of Dermatology; 146; 886-890 Schnuch A, Lessmann H, Geier J, Uter W (2011). Contact allergy to preservatives. Analysis of IVDK data 1996-2009. The British Journal of Dermatology; 164; 13161325 Ackermann L, Aalto-Korte K, Alanko K, Hasan T, Jolanki R, Lammintausta K, Lauerma A, Laukkanen A, Liippo J, Riekki R, Vuorela AM, Rantanen T (2011). Contact sensitization to methylisothiazolinone in Finland--a multicentre study. Contact Dermatitis; 64; 49-53 Lundov MD, Zachariae C, Johansen JD (2011). Methylisothiazolinone contact allergy and dose-response relationships. Contact Dermatitis; 64; 330-336 Lundov MD, Krongaard T, Menné TL, Johansen JD (2011). Methylisothiazolinone contact allergy: a review. The British Journal of Dermatology; 165; 1178-1182 Uter W, Werner A, Armario-Hita JC, Fernandez-Vozmediano JM, Ayala F, Balato A, Bauer A, Ballmer-Weber B, Beliauskiene A, Fortina AB, Bircher A, Brasch J, Chowdhury MM, Coenraads PJ, Schuttelaar ML, Cooper S, Czarnecka-Operacz M, Zmudzinska M, Elsner P, English JS, Frosch PJ, Fuchs T, García-Gavín J, Fernández-Redondo V, Gawkrodger DJ, Giménez-Arnau A, Green CM, Horne HL, Johansen JD, Jolanki R, Pesonen M, King CM, Krêcisz B, Chomiczewska D, Kiec-Swierczynska M, Larese F, Mahler V, Ormerod AD, Peserico A, Rantanen T, Rustemeyer T, Sánchez-Pérez J, Sansom JE, Silvestre JF, Simon D, Spiewak R, Statham BN, Stone N, Wilkinson M, 31 SCCS/1482/12 Opinion on benzisothiazolinone ___________________________________________________________________________________________ 60. 61. Schnuch A (2012). Current patch test results with the European baseline series and extensions to it from the 'European Surveillance System on Contact Allergy' network, 2007-2008. Contact Dermatitis; 67; 9-19 Uter W, Gefeller O, Geier J, Schnuch A. (2012). Methylchloroisothiazolinone/methylisothiazolinone contact sensitization: diverging trends in subgroups of IVDK patients in a period of 19 years. Contact Dermatitis; Epub ahead of print 5 May 2012; doi: 10.1111/j.1600-0536.2012.02075.x. Maio P, Carvalho R, Amaro C, Santos R, Cardoso (2012). Contact allergy to methylchoroisothiazolinone/methylisothiazolinone (MCI/MI): findings from a Contact Dermatitis Unit. J Cutaneous and Ocular Toxicology; 31; 151-153 32
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