1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 July 2017 - 21 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Not applicable

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

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

210420-85-2

Constituent 2

Reference substance name:

N-methyldidecylamine

EC Number:

230-990-1

EC Name:

N-methyldidecylamine

Cas Number:

7396-58-9

Molecular formula:

C21H45N

IUPAC Name:

N-decyl-N-methyldecan-1-amine

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

impurity 2

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 3

Reference substance name:

Adipic acid

EC Number:

204-673-3

EC Name:

Adipic acid

Cas Number:

124-04-9

Molecular formula:

C6H10O4

IUPAC Name:

adipic acid

Test material form:

solid

Details on test material:

- Appearance: Slightly yellow gel
- Storage conditions: At room temperature; container flushed with nitrogen

Specific details on test material used for the study:

not applicable

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on animal used as source of test system:

not applicable

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 26490 (kit K and L) and 26708 (kit L and M)
- Surface: 0.6 cm^2
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure: room temperature, 1 hour exposure: 36.8 - 37.5°C.
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed with phosphate buffered saline (one washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Amount of MTT medium used: 300 µL
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 4 (2 tissues for the 3 minute exposure period, 2 tissues for the 1 hour exposure period) + 2 tissue for the negative and the positive control for each exposure period

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

PREDICTION MODEL / DECISION CRITERIA (see table 1 in ''any other informatin on methods'')
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability = 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit =0.8 and upper acceptance limit =2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be = 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: an excess amount (undiluted)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL (8N KOH)

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

4 in total; 2 per exposure period

Test animals

Details on test animals or test system and environmental conditions:

not applicable

Test system

Type of coverage:

open

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Liquid test item applied directly on top of the tissue

Duration of treatment / exposure:

3 minute exposure and 1 hour exposure

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

not applicable

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 2 replicates

Run / experiment:

3-minute exposure period; experiment 1

Value:

53

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

6.7%

Remarks on result:

other: CV between replicates: 16%

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 2 replicates

Run / experiment:

3-minute exposure period; experiment 2

Value:

39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

6.3%

Remarks on result:

other: CV between replicates: 10%

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 2 replicates

Run / experiment:

1-hour exposure period

Value:

36

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

9.6%

Remarks on result:

other: CV between replicates: 23%

Other effects / acceptance of results:

- The individual viabilities of the tissues treated with test item were spread over 2 categories (48% and 57%) therefore the 3-minute exposure period was repeated. The repeated experiment showed individual tissue viabilities of 37% and 42% and was therefore considered acceptable and used to determine the final conclusion.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 was between the acceptance limits (i.e., 1.971 for the 3-minute exposure and 1.744 for the 1-hour exposure).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1 hour exposure was <15% (i.e., 9.6%).
- Acceptance criteria met for variability between replicate measurements: yes, CV between tissue replicates was =30% (i.e., 10% for the 3 minute exposure periods and 23% for the 1 hour exposure period).

In vivo

Irritant / corrosive response data:

not applicable

Other effects:

not applicable

Any other information on results incl. tables

Table 2 Individual OD measurements at 570 nm

	3-minute application (OD570)        A               B		1-hour application (OD570)       A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.6481	1.7265	1.7199	1.8792
	1.6399	1.7571	1.6803	1.8309
	1.7038	1.7773	1.7232	1.8794
1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.8198	0.9787	0.5830	0.7528
	0.8592	1.0003	0.5961	0.7578
	0.8449	1.0043	0.6021	0.7686
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.1488	0.1510	0.2237	0.1978
	0.1585	0.1482	0.2186	0.1980
	0.1607	0.1536	0.2237	0.1954

Repeat experiment	3-minute application (OD570)        A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.9161	2.0235
	2.0228	2.0685
	2.0073	2.0446
1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.7352	0.8733
	0.7976	0.8560
	0.8016	0.8579
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.1424	0.1780
	0.1579	0.1833
	0.1567	0.1826

OD = Optical density; Duplicate exposures are indicated by A and B.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The results of a skin corrosion test, performed according to OECD guideline 431, showed that 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) was corrosive to the skin (tissue viability of 39% after 3-minute exposure). The test substance is therefore classified as category 1 according to GHS and according to Regulation (EC) 1272/2008.

Executive summary:

The results of a skin corrosion test, performed according to OECD guideline 431, showed that 1-Decanaminium, N-decyl-N,N-dimethyl-, hexanedioate (2:1) was corrosive to the skin (tissue viability of 39% after 3-minute exposure). The test substance is therefore classified as category 1 according to GHS and according to Regulation (EC) 1272/2008.