Opinion Of The Scientific Committee On Consumer Safety Benzisothiazoli P96 Sccs O 099

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ACKNOWLEDGMENTS
1. BACKGROUND
2. TERMS OF REFERENCE
3. OPINION
4. CONCLUSION
5. MINORITY OPINION
6. REFERENCES

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

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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

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

SCCS/1482/12

Opinion on benzisothiazolinone

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ACKNOWLEDGMENTS

Prof. J. Angerer

Dr. U. Bernauer

Dr. C. Chambers

Prof. G. Degen (rapporteur)

Dr. W. Lilienblum (associate scientific advisor)

Dr. E. Nielsen

Dr. S.C. Rastogi

Prof. V. Rogiers

Prof. T. Sanner (chairman)

Dr. J. van Engelen

Prof. R. Waring

Dr. I.R. White

Keywords: SCCS, scientific opinion, preservative, benzisothiazolinone, P96, directive

76/768/ECC, CAS 2634-33-5, EC 220-120-9

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.

SCCS/1482/12

Opinion on benzisothiazolinone

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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. Chemical identity........................................................................6

3.1.2. Physical form .............................................................................6

3.1.3. Molecular weight ........................................................................6

3.1.4. Purity, composition and substance codes........................................ 7

3.1.5. Impurities / accompanying contaminants .......................................7

3.1.6. Solubility ...................................................................................7

3.1.7. Partition coefficient (Log Pow) ......................................................8

3.1.8. Additional physical and chemical specifications................................8

3.1.9. Homogeneity and Stability ...........................................................8

3.2. Function and uses................................................................................ 8

3.3. Toxicological Evaluation ........................................................................ 9

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

4. CONCLUSION ............................................................................................ 28

5. MINORITY OPINION.................................................................................... 28

6. REFERENCES............................................................................................. 29

SCCS/1482/12

Opinion on benzisothiazolinone

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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?

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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: 2634-33-5

EC: 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

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3.1.4. Purity, composition and substance codes

Substance code: /

Batches used: batch no 2001 014

Purity: 74.02-84.02% w/w (84% corresponds to 15% water content)

> 99% w/w on a dry basis

Loss on drying: /

Water content: 15-29% w/w (for batch no 2001 014 spec., 20% max, found 15%)

Ash content: /

Sodium chloride: < 0.1% w/w (for batch no 2001 014 spec., 0.2% max, found 0.02%)

Lead: /

Mercury: /

Arsenic: /

Iron: /

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

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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 Pow: 0.99 at 20 °C and pH 5

Log Pow: 0.63 at 10 °C and at pH 7

Log Pow: 0.70 at 20 °C and pH 7

Log Pow: 0.76 at 30 °C and pH 7

Log Pow: – 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: 156.6 °C (Directive 92/69/EEC, A1)

Boiling point: 327.6 °C (Directive 84/449/EEC, A2)

Density: 1.483 g/cm³ at 20 °C (OECD Guideline 109)

Vapour Pressure: 0.0000037 hPa at 25 °C (Directive 92/69/EEC, A4)

pKa: 7.3 at 25 °C

Flash point: /

Viscosity: /

Refractive index: /

UV_Vis spectrum (200-800 nm): /

3.1.9. 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:

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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: EPA OPP 81-1

Species/strain: Rat, Sprague-Dawley derived, albino

Group size: 30 (3 groups of 5 males and 5 females each)

Test item: Benzisothiazolinone

Test substance: Nuosept BIT Technical

Batch: # 170-138 (PSL Code no E50629-1R (powder)

Purity: 1,2-Benzisothiazolin-3-one 82.3%; water 17.7%

Dose: 1000, 2000 and 5000 mg/kg bw/day test substance (823, 1646 and

4115 mg/kg active ingredient)

Vehicle: Water; the test substance was applied as 40% w/w suspension in water

Route: oral intubation/gavage

GLP: 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

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3.3.1.2. Acute dermal toxicity

Taken from SCCNFP/08441/04

Guideline: /

Method: EPA OPP 81-2

Species/strain: rat, Sprague-Dawley derived, albino

Group size: 10 (5 male/5 female)

Test item: benzisothiazolinone

Test substance: nuosept BIT Technical

Batch: # 170-138 (PSL Code no E50629-1R (powder)

Purity: 1,2-Benzisothiazolin-3-one 82.3%; water 17.7%

Dose: 5000 mg/kg bw/day (Limit test) (4115 mg/kg active ingredient)

Vehicle: water; the test substance was moistened with water for application (1

ml water/1 g test substance)

Route: topical application (24 h)

Exposure period: 1 x 24 h. observation period 14 d

GLP: 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: EPA OPP 81-5

Species/strain: New Zealand albino rabbits

Group size: 6, 3 males and 3 females

Test substance: Nuosept BIT Technical

Batch: # 170-138

Purity: 1,2-Benzisothiazolin-3-one, 82.3%; Water, 17.7%

Dose: Slurry of 0.5 g in 0.5 ml water

Exposure: Semi occlusive

Exposure time: 4 hour(s)

Readings: 1, 24, 48, 72 hours and 7 days

GLP: in compliance

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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: EPA OPP 81-4

Species/strain: New Zealand albino rabbits

Group size: 9 (4 males and 5 females)

Test substance: Nuosept BIT Technical

Batch: # 170-138

Purity: 1,2-Benzisothiazolin-3-one, 82.3%; Water, 17.7%

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Dose: 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.

Exposure time: 48 hours

GLP: in compliance

Date: 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: INVITTOX Protocol 124

Species: Bovine

Number corneas: 45

Test substance: 1,2-Benzisothiazolin-3-one

Batch: LHW 1355

Purity: >99%

Dose: 75, 750 and 7500 ppm aqueous solution of BIT prepared as its sodium

salt

Exposure time: 10 minutes

GLP: in compliance

Date: 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.

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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: BALB/c 3T3 Mouse fibroblast cell line

SIRC Rabbit corneal cell line

Test substance: 1,2-Benzisothiazolin-3-one

Batch: LHW 1355

Purity: > 99%

Dose: 0-100 ppm range finding; 0-10 ppm testing

Exposure time: 24 hours

GLP: in compliance

Date: 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 EC50

3T3

EC50 SIRC

Chloromethylisothiazolinone/methylisothiazolinone (3:1) 1.19 1.89

Methylisothiazolinone 5.76 5.98

Methyldibromoglutaronitrile 20% (in phenoxyethanol) 28.4 29.36

Methyl/ethyl/propyl/butyl/isobutyl parabens in phenoxyethanol 439.5 489.0

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: OECD 406

Species/ strain: Albino Dunkin Hartley guinea pigs

Size: 38 (20 test, 10 control, 8 range-finding)

Test substance: 1,2-benzisothiazolin-3-one 79.8%, water 19.2%

Batch: 386-3

Purity: 79.8% 1,2-benzisothiazolin-3-one (BIT), water, 19.2%

Diamide content 0.28%, PCP < 1 ppm.

Dosage: 1st induction 0.1% w/v intracutaneous

nd induction 20% w/v occlusive epicutaneous

rd challenge 10% w/v occlusive epicutaneous

Vehicle: corn seed oil (1st and 2nd concentration), FCA/water (1st

concentration), Ethanol (3rd concentration)

GLP: in compliance

Date: 1996

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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: EPA OPP 81-6

Species/strain: Hartley albino guinea pigs

Size: 8 for range finding, 30 for test protocol

Test substance: Nuosept Bit technical (82.3% 1,2-Benzisothiazolin-3-one, water 17.7%)

Batch: #170-138

Purity: 1,2-Benzisothiazolin-3-one (BIT) 82.3% a.i.

Dosage: 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

Vehicle: Corn seed oil

GLP: in compliance

Date: 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 (dichloronitro-

benzene) 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 chloromethyliso-

thiazolinone.

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.

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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.

Product Code Assessment grading Product

Code 1 2

Withdrawn 3 3

No Assessment 2 2

No Visible Redness 9 10

Slight Redness 0 0

Distinct Redness 1 0

Total 15 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 Test concentration

(ppm)

Proportion of human

panel sensitised (%)

LLNA EC3

BIT 725 5/58 (9%) 10.4%

BIT 360 0/54 (0%)

MTI 300 3/19 (16%) 2.0%

MTI 100 0/211(0%)

CIT/MIT 20 2/45 (4.4%) 0.007-0.01%

CIT/MIT 10 0/175(0%)

Ref.: 24

Comment

The HRIPT is not considered ethical.

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Local Lymph Node Assay

An EC3 of 2.3% for 1,2-benzisothiazolin-3-one (CAS 2634-33-5) and 1.9% for 2-methyl-

2H-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: OECD 428

Species/strain: human skin (5 abdominal, 1 breast); 400 μm dermatomed skin

Group size: 10 membranes

Method flow through diffusion cell

Membrane integrity Tritiated water

Test substance: benzisothiazolinone

Batch: 12409EE

Purity: 97%

Radiochemical 14C benzisothiazolinone; 99.9%

Vehicle: water

Test item: 0.01% w/v benzisothiazolinone aqueous

Dose volume: 20 μl/cm2

Receptor Tissue culture medium containing 6% PEG, 0.01% sodium azide,

1% glucose and streptomycin

Solubility (in water) 1 mg/ml

Method of Analysis: liquid scintillation

GLP: in compliance

Study period: 2008

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The absorption of radiolabelled 0.01% w/v benzisothiazolinone in aqueous solution was

determined from the use of 10 chambers with human dermatomed skin.

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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: OECD 407

Species/strain: Rat, Wistar Hsd Cpb:WU

Group size: 12 (6 male/6 female)

Test item: Benzisothiazolinone, Code 072/1-PBP

Test substance: Promex BIT (paste)

Purity: 1,2-Benzisothiazolin-3-one, 84.29%; water, 15%, purity of active

ingredient on dry weight basis, 99.02%

Batch: 2001 014//sample no. KP 070601//cb 181100

Dose levels: 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

Route: daily oral intubation/gavage

Exposure period: 28 days

GLP: in compliance

Date: 2001

Results

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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: OECD 408

Species/strain: Rat, Wistar

Group size: Total 20; 10 male/10 female

Test item: Benzisothiazolinone (Promex-BIT)

Batch: G00Z-0600-1907-13//072/1.PBP//2001 014//KP 070601

Purity: 1,2-Benzisothiazolin-3-one, 84.29%; water 15%; purity of active

ingredient on dry weight base 99.1%

Dose levels: 10, 30 and 75 mg/kg bw/day (8.42, 25.26 and 63.15 mg/kg bw/day

a.i.)

Vehicle: 0.5% carboxymethylcellulose (CMC)

Route: daily oral intubation/gavage

Exposure period: 90 days

GLP: in compliance

Study period: 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.

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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 non-

glandular 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 non-

glandular 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 (hyperkera-

tosis, 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: OECD 471 (1997)

Species/strain: Salmonella typhimurium TA98, TA1537, TA100, TA1535

Escherichia coli WP2uvrA pkM 101

Test substance: Promex BIT 1,2–Benzisothiazolin-3-one

Batch: 2001 014

Lot number: KP 070601

Purity: 99.02%

Concentrations: 0, 20, 35, 60, 100 and 175 μg/plate (1st experiment)

0, 30, 50, 75, 120 and 180 μg/plate (2nd experiment)

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Replicate: 3 plates/concentration

Positive controls: according to guideline

Metabolic activation: Aroclor induced rat liver homogenate

GLP: in compliance

Date: 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: OECD 476 (1997)

Species/strain: CHO-K1 (Chinese hamster ovary cells) HPRT locus

Test substance: Promex BIT; 1,2-Benzisothiazolin-3-one

Batch: 2001 014

Lot number: KP 070601

Purity: 99.02%

Concentrations: 0, 0.65, 1.30, 2.60, 5.20 μg/ml

Treatment time: 5 hours, with and without metabolic activation

Replicate: 2 experiments in the same conditions.

Positive controls: B(a)P; EMS

Metabolic activation: Aroclor 1254 induced rat liver homogenate.

GLP: in compliance

Date: 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: OECD 473 (1997)

Species/strain: CHO-K1 cell line (Chinese hamster ovary cells)

Test substance: Promex BIT; 1,2–Benzisothiazolin-3-one

Batch: 2001 014

Lot number: KP 070601

Purity: 99.02%

Concentrations: 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

Replicate: 2 experiments (200 metaphases analysed)

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Treatment time: 1st experiment: 3 hours

2nd experiment: 3 hours, in the presence of S9-mix; 19 hours, in

the absence of S9-mix

Positive controls: CPA (55 μg/ml); EMS (600 μg/ml)

Metabolic activation: Aroclor 1254 induced rat live homogenate

GLP: in compliance

Date: 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: OECD 474 (1997)

Species/strain: Swiss albino mice-HsdOla: MF1 strain

Test substance: Promex BIT; 1,2–Benzisothiazolin-3-one

Batch: 2001 014

Lot number: KP 070601

Purity: 99.02%

Doses: 63.15; 126.3; 210.5 mg/kg a.i.

Treatment: oral (gavage) twice, at 24 hours of interval. The animals were sacrificed

24 hours after the second treatment.

Positive control: CPA, 40 mg/kg bw, oral treatment

GLP: in compliance

Date: 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: OECD 486 (1997)

Species/strain: Wistar Hanlbm: WIST (SPF) rats

Test substance: Promex BIT

Batch: 2001014

Purity: 99.02%

Doses: 0 (corn oil), 375, 750 mg a.i./kg bw

Treatment times: 2 hours, 16 hours, orally, once

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Positive controls: N,N’-dimethylhydrazine dihidrochloride (DMH): 40 mg/kg bw; 2 hours 2-

acetylaminofluorene (2-AAF): 100 mg/kg bw; 16 hours.

GLP: in compliance

Date: 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: EPA Health Effects Test Guidelines OPPTS 870.3800, of aug 1998

Species/strain: Crl:W1 (Glx/BRL/Han)BR

Group size: 24 males and 24 females (P generation)

Test substance: Proxel Press Paste (1,2-Benzisothiazolin-3-one)

Batch: No. 103 and No. 344

Purity: Purity of active ingredient: 92.7% +/- 1.1%

Dose levels: P generation: 250, 500 and 1000 ppm

F1 generation: 250, 500 1000 ppm

Route: oral

Administration: dietary

Control: control diet only

GLP statement: Yes

Study period: 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

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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 pre-

pairing 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

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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

Ref.: 14

3.3.9. Toxicokinetics

No data provided.

3.3.10. Photo-induced toxicity

3.3.10.1. Phototoxicity / photoirritation and photosensitisation

Guideline: OECD Test guideline 432

Test: Neutral Red uptake phototoxicity test with Balb/c 3T3 cells

Test substance: 1,2–Benzisothiazolin-3-one

Batch: LMS 414, CH-0405-ST-7

Purity: > 99%

UV-A Irradiation: SOL-500 lamp fitted with a H1-filter mounted at a distance of 60 cm

to achieve an UV-irradiance of 5 J/cm2

Concentrations: 0, eight concentrations between 0.2 to 50 µg/mL in the presence and

absence of UV-A exposure

Replicate: 3 main experiments

Positive control: Chlorpromazine

GLP: in compliance

Date: 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

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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) = 17.4 g/d

Concentration Benzisothiazolinone (BIT) = 0.01%

Daily exposure BIT = 1.74 mg

Dermal absorption = 61.9%

Typical body weight of human = 60 kg

Systemic exposure dose = 0.018 mg/kg bw/d

No Observed Adverse Effect Level = 50 mg/kg bw/d

(2-generation-study, oral, rat)

NOAEL corrected for 50% oral bioavailability = 25 mg/kg bw/d

Margin of Safety NOAEL/SED = 1392

3.3.14. 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 chloromethyl-

isothiazolinone 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

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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 28-

day and 90-day studies were based on histopathological lesions observed in the non-

glandular 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 non-

glandular 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

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

SCCS/1482/12

Opinion on benzisothiazolinone

___________________________________________________________________________________________

6. REFERENCES

1. Material Safety Data Sheet, Acticide BIT, Thor GmbH 22-11-2001

2. 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

3. 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

4. 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

5. RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702696

"Determination of the Vapour Pressure of ACTICIDE BIT" (Sponsor: THOR Chemie

GmbH), 19-11-1998

6. 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

7. 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

8. RCC Ltd, Environmental Chemistry & Pharmanalytics Div., Project No. 702431

"Determination of the Water Solubility of ACTICIDE BIT" (Sponsor: THOR Chemie

GmbH), 19-11-1998

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. Thor Specialities (UK) Limited, Study RS/02/036. “3T3 Neutral Red Uptake

Phototoxicity assay “. (Sponsor: Thor Group Management Limited) March 2005.

17. 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

18. 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.

19. 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

20. 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

SCCS/1482/12

Opinion on benzisothiazolinone

___________________________________________________________________________________________

21. psl - product safety labs, Study No. 3863 "Dermal sensitization test – Buehler Method"

(Sponsor: Hüls America Inc., joint ownership: Prom Chem Ltd and Thor GmbH), 24-

01-1996

22. 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.

23. 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

24. 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.

25. Toxicology Department Rallis Research Centre, Study No. 3288/01 "Bacterial Reverse

Mutation Test with Promex BIT" (Sponsors: Prom Chem Ltd and Thor GmbH), 01-03-

26. 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

27. 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

28. 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

29. RCC Cytotest Cell Research. Study Number: 736501. "Unscheduled DNA synthesis with

1,2-Benzisothiazolin-3-one“ Study Commissioned June 2002

30. B.R Alexander (2002) An assessment of the comparative sensitisation potential of

some common isothiazolinones. Contact Dermatitis 46, 191-196

31. Steinberg, D.C. (2000) Measuring synergy. Cosmetics & Toiletries 115(11), 59-62.

32. 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.

33. Seal K.J., Alexander B.R., (2001) Special Agents. Global Cosmetic Industry, 169 (6),

24-30

34. Chapman, J.S., Diehl, M.A., Fearnside, K.B. (1998) Preservative tolerance &

resistance. International Journal of Cosmetic Science 20, 31-39.

35. 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

36. 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. Ayadi M, Martin P. Pulpitis of the fingers from a shoe glue containing 1,2-

benzisothiazolin-3-one. Contact Dermatitis 1999; 40: 115-116

38. 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

39. Taran JM, Delaney TA. Allergic contact dermatitis to 1,2-benzisothiazolin-3-one in the

carpet industry. Australas J Dermatol 1997; 38: 42-43

40. 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

SCCS/1482/12

Opinion on benzisothiazolinone

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41. 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.

42. Burden AD, O’Driscoll JB, Page FC, Beck MH (1994) Contact hypersensitivity to a new

isothiazolinone. Contact Dermatitis 30; 179-180

43. 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

44. Dias M, Lamarao P, Vale T (1992) Occupational contact allergy to 1,2-benzisothiazol-3-

one in the manufacture of air fresheners. Contact Dermatitis 36; 205-206

45. Greig DE (1991) Another isothiazolinone source. Contact Dermatitis 25; 201-202

46. 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.

47. 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.

48. 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.

49. 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; 1019-

1030.

50. 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

51. García-Gavín J, Goossens A (2010). Moist toilet paper: allergy to the nonhalogenated

derivative methylisothiazolinone preservative alone. Archives of Dermatology; 146;

1186

52. Amaro C, Santos R, Cardoso J (2011). Contact allergy to methylisothiazolinone in a

deodorant. Contact Dermatitis; 64; 298-299

53. Lundov MD, Thyssen JP, Zachariae C, Johansen JD (2010). Prevalence and cause of

methylisothiazolinone contact allergy. Contact Dermatitis; 63; 164-167

54. 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

55. 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; 1316-

1325

56. 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

57. Lundov MD, Zachariae C, Johansen JD (2011). Methylisothiazolinone contact allergy

and dose-response relationships. Contact Dermatitis; 64; 330-336

58. Lundov MD, Krongaard T, Menné TL, Johansen JD (2011). Methylisothiazolinone

contact allergy: a review. The British Journal of Dermatology; 165; 1178-1182

59. Uter W, Werner A, Armario-Hita JC, Fernandez-Vozmediano JM, Ayala F, Balato A,

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Schnuch A (2012). Current patch test results with the European baseline series and

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60. Uter W, Gefeller O, Geier J, Schnuch A. (2012).

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Opinion Of The Scientific Committee On Consumer Safety Benzisothiazoli P96 Sccs O 099

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