Reaction mass of 2-amino-2-methyl-1-propanol hydrochlorid (3207-12-3) and 2-amino-2-methyl-1-propanol (124-68-5)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 February 2018 - 07 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

Please refer to the "Principles of method if other than guideline" field.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2017

Deviations:

yes

Remarks:

Please refer to the "Principles of method if other than guideline" field.

Principles of method if other than guideline:

The pre-incubation period was 16 h (guideline requirement: 18-24 h) and the post-incubation period was 40 h 24 min (guideline requirement: 42 h). The incubation periods used are in accordance with the validated MatTek protocol. This deviation does not affect the validity or quality of the result. The test item is considered to be classified as irritant or corrosive (UN GHS Cat. 2 or Cat 1). A longer pre- and post-incubation period is expect to reveal the same result.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

EC Number:

221-713-5

EC Name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

Cas Number:

3207-12-3

Molecular formula:

C4H11NO.ClH

IUPAC Name:

1-hydroxy-2-methylpropan-2-aminium chloride

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT)
- Tissue batch number: 25891

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 15 times filling and emptying the tissue inserts with DPBS, 3 times shaking with approx. 50 mL DPBS
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The tissue viability was within the acceptable range.
- Barrier function: The barrier function was within the acceptable range.
- Contamination: No contaminations were determined.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the viability after exposure and post-exposure is less than or equal to 50 %.
- The test substance is considered to be non-corrosive and non-irritant to skin if the viability after exposure and post-exposure is more than 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% SDS

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

22 h 58 min

Number of replicates:

3 replicates per test item, 3 replicates negative controls and 3 replicates positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1-3

Value:

17.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Summary of optical desity (OD) and Viability (%)

Treatment		OD	Viability (%)
Negative Control (DPBS)	Mean	1.879	100
	±SD	0.075	4.01
	n	3	3
Positive Control (5% Sodium Dodecyl Sulphate)	Mean	0.066	3.5
	±SD	0.003	0.15
	n	3	3
Test Item	Mean	0.322	17.2
	±SD	0.028	1.51
	n	3	3

n = No. of tissues; SD = Standard Deviation

    

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (corrosive) or Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control.

Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using Reconstructed Human Epidermal Model-EpiDerm™ (EPI-200-SIT) according to OECD Guideline for the testing of chemicals No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”, adopted on 28thJuly 2015. The test item did not develop any colour when dissolved in distilled water or isopropanol and was considered not to reduce MTT as no purple colour was developed when mixed and incubated with MTT solution. After receipt of the tissues, visual inspection was performed. As there were no tissue defects, air bubble or excess moisture observed, all the tissue inserts were used for the study.Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO₂ incubator for 55 minutes. After 55 minutes of incubation, tissues were transferred to lower row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO₂ incubator for 15 hours. After the incubation period, the tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 30 µL of the test item. All treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator for 22 hours and 58 minutes. After incubation period (Day 1), tissue inserts were shifted from upper wells to lower wells of 6 -well plates prefilled with 0.9 mL of assay medium. After the media change, tissues were incubated for an additional 17 hours and 26 minutes in CO2 incubator. After post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours using extraction solvent (MTT-EXT-100). The optical density of the extracted formazan was measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated by entering OD values in the spread sheet provided by MatTek.

Percentage viability of negative control, positive control and test item was 100±4.01 %, 3.5±0.15 % and 17.2±1.51 % respectively. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritating/corrosive potential of the positive control and the suitability of the test method. As the percentage viability of the test item was less than 50% of the negative control, the test item is considered to be irritant or corrosive to skin (UN GHS Category 2 or Category 1).