Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-16 to 2017-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

IUPAC Name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Test material form:

other: solid, very viscous yellow paste

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm.
- Pre-incubation: 60 minutes in the incubator (37 ± 1 °C, 5 ± 0.5% CO2) in the upper wells. Then transferred from upper wells into the lower wells for about 19 hours (37 ± 1 °C, 5 ± 0.5% CO2).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- per replicate: 25.2, 25.8 and 24.5 mg, respectively

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

23.5 h

Number of replicates:

3

Test system

Details on study design:

Details of the test procedure used
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (EPI-212-SIT)
- Conditions of exposure: 37 ± 1 °C, 5 ± 0.5% CO2
- Washing: inserts gently rinsed with DPBS
- Number of tissue replicates used per test chemical and controls: 2
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes at 37 ± 1 °C, 5 ± 0.5% CO2)
- Data evaluation: the following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. The mean of the photometric absorbance of the negative control is set to 100%. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability(%) = (mean OD test item / positive control / mean OD negative control) x 100. For the test item and the positive control themean relative viability ± rel. standard deviation of the three individual tissues was calculated
- Description of evaluation criteria: For the current test, an irritation potential of the test item of H315, GHS Cat 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
- Historical data positive control: Mean Viability:5.1%; Rel. Standard Deviation: 3.4%; Range of Viabilities: 2.0 - 17.1%.
- Historical data negative control: Mean Absorption: 1.854%; Rel. Standard Deviation: 0.336%; Range of Absorbance: 0.476 - 2.471
- Acceptability of the Assay: the results are acceptable if (1) tissue viability is meeting the acceptance criterion, i.e. if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 (negative control). If (2) the relative tissue viability of the positive control is ≤ 20% (positive control). If (3) the SD of 3
identical replicates is < 18%. OD values should not be below historically established boundaries.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

< 5.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The acceptance criteria were met.

Any other information on results incl. tables

The mean absorbance values measured for the 3 tissues (mean - blank) is presented below:

Sample	Negative control	Positive control	Test item
Tissue 1	2.023	0.040	0.120
Tissue 2	2.044	0.033	0.111
Tissue 3	2.047	0.040	0.119
Mean of 3 tissues	2.038	0.038	0.117

This resulted in following % viability:

Sample	Positive control	Test item
Tissue 1	2.0%	5.9%
Tissue 2	1.6%	5.4%
Tissue 3	2.0%	5.8%
Mean of 3 tissues	1.8%	5.7%
SD of mean viability	0.2%	0.2%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item is considered as irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.

Executive summary:

In an in vitro study the skin irritation potential of the test item was assessed with the Reconstructed Human Epidermis (RhE) Test Method according to EU-Method B.46 resp. OECD 439 and GLP. In this study the test item was applied on top of a human reconstructed epidermis model. The cell viability was measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide] into a blue formazan salt that was quantitatively measured (as optical density (OD)) after extraction from the tissues.

In the pre-tests, neither direct MTT reduction by the test item, nor a possible binding capacity of the test item could be observed. Therefore, no additional tests were necessary.

In the main test, three tissues of the human skin model EpiDermTM were treated with the test substance, the negative control (DPBS), or the positive control (5% SDS) for 60 minutes. The test item, the negative control, and the positive control were applied directly on top of each tissue and spread to match the tissue size (0.63 cm2).

After treatment with the negative control, the absorbance value (OD was 2.0) was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, thus showing the quality of the tissues. The positive control induced a decrease to 1.8% of the relative absorbance compared to the negative control, indicating clear irritating effects, and thus ensuring the validity of the test system. The variation within the tissue replicates was below the threshold of the OECD TG (<18%), thus ensuring the validity of the study.

After the treatment with the test item, the mean relative absorbance value was reduced to 5.7 % compared to the negative control. This value is below the threshold for skin irritancy of 50% and therefore the test item is considered to possess skin irritant potential.