2-methyl-2H-isothiazol-3-one hydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2017 to 21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

(2016)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-Methyl-4-Isothiazolin-3-One Hydrochloride

Cas Number:

26172-54-3

Molecular formula:

C4H5NOS.HCl

IUPAC Name:

2-Methyl-4-Isothiazolin-3-One Hydrochloride

Test material form:

solid

Details on test material:

MIT.HCl is manufactred as >99.9% purity therefore technical grade and anlaytical grade material are identical

Specific details on test material used for the study:

- Identification: MIT.HCl (Methyl isothiazolinone hydrochloric acid)
- CAS no.: 26172-54-3
- Source and lot/batch No.of test material: Sigma-Aldrich / 10013913
- Expiration date of the lot/batch: 30 Nov 2018
- Purity test date: 23 Nov 2016
- Purity: >99.9%
- Apperance: White to light beige powder
- Moelcular weight: 151.61 g/mol
- Storage condition of test material: 2 to 8°C, protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

other: Normal human epidermal keratinocytes (NHEK), derived from neonatal-foreskin tissue

Details on animal used as source of test system:

TEST KIT
- Source: MatTex Corporation
- Type: Three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum

Cell culture media:
- Assay medium (MatTex Corporation)
- Vehicle / postive controls: purifed water / 8N KOH

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37°C, humidified incubator
- CO2 (%): 5%

IN-LIFE DATES: 20 September 2017 to 21 September 2017

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-experimental checks:
To check the non-specific MTT-reducing capability of the test article 25 mg of the test article was mixed with 1 mL MTT (1 mg MTT/mL) medium and incubated for 1 hour and observed visually after stirring.

Assessment for Colour Interference
25 mg of test article was added to 0.3 mL of both deionized water and isopropanol and incubated for 60 minutes at 37°C, 5% CO2, 95% RH. Neither solution became coloured, therefore it was deemed not to have the potential to stain the tissue.

Control samples:

yes, concurrent vehicle

Amount/concentration applied:

50 uL of test article

Duration of treatment / exposure:

single exposure / 3 and 60 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

2 replicates/dose/time point

Test animals

Details on test animals or test system and environmental conditions:

n/a, in vitro test system used

Test system

Amount / concentration applied:

n/a, in vitro test system used

Duration of treatment / exposure:

n/a, in vitro test system used

Observation period:

n/a, in vitro test system used

Number of animals:

n/a, in vitro test system used

Details on study design:

n/a, in vitro test system used

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive indication of corrosivity

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 5.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: positive indication of corrosivity

Other effects / acceptance of results:

refer to "Any other information on results incl. tables"

Any other information on results incl. tables

Table 7.3.1/01-1: Summary of in vitro EPIDERM corrosivity result following application of MIT.HCL

Test Substance	Mean OD 579	Mean relative tissue viability (%) ±CV
3 minute exposure
Negative	1.247	100 ±1.7
Test article	0.269	22 ±4.9
Positive	0.319	-6 ±0.8
Positive FK	0.391
60 minute exposure
Negative	1.260	100 ±2.4
Test article	0.071	5.6 ±6.5
Positive	0.168	-0.1 ±2.6
Positive FK	0.170

FK          Freeze killed (Positive control tissue mean)

 

Skin viability after a three minute or one hour exposure to the test article was 22% and 5.6%, respectively.

The OD values for the negative controls met the acceptance criteria.

 

Skin viability after a three minute or one hour exposure to the positive control article was -6% and -0.1%, respectively, demonstrating appropriate performance of the assay.

 

All CVs between replicates were less than 30% and met the acceptance criteria.

 

The negative and positive control values fell within the laboratory historical control values.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Conclusions:

Under the conditions of this study the MIT.HCl showed irritant effects. The mean relative tissue viability was <50% after 3 minute (22%) and 1 hour (5.6%) exposure. Therefore test article, MIT.HCl, was considered to be corrosive (Sub-category 1A) according to the UN GHS classification system

Executive summary:

This study was conducted to determine whether the test article,MIT.HCl, causes corrosion in thein vitroskin model EpiDerm^TM.

 

Duplicate EpiDerm^TM inserts were treated with test article, purified water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 1 hour. At the end of the treatment period, the tissues were washed with phosphate buffered saline (PBS) and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times.

 

Skin viability after a three minute or one hour exposure to the test article was 22% and 5.6%, respectively.

The OD values for the negative controls met the acceptance criteria.

 

Skin viability after a three minute or one hour exposure to the positive control article was -6% and -0.1%, respectively, demonstrating appropriate performance of the assay.

 

All CVs between replicates were less than 30% and met the acceptance criteria.

 

The negative and positive control values fell within the laboratory historical control values.

 

Under the conditions of this study the MIT.HCl showed irritant effects. The mean relative tissue viability was <50% after 3 minute (22%) and 1 hour (5.6%) exposure. Therefore test article, MIT.HCl, was considered to be corrosive (Sub-category 1A) according to the UN GHS classification system.