nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2016 - 28 November 2016

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)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

EC Number:

946-436-3

Molecular formula:

UVCB

IUPAC Name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

impurity 1

Reference substance name:

(p-chlorophenoxy)isobutyroyl chloride

EC Number:

255-286-1

EC Name:

(p-chlorophenoxy)isobutyroyl chloride

Cas Number:

41267-93-0

Molecular formula:

C10H10Cl2O2

IUPAC Name:

2-(4-chlorophenoxy)-2-methylpropanoyl chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid

Details on test material:

Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-047
- The skin was moistened with 5 μL Milli-Q water to ensure close contact of the test item to the tissue and the solid test item (26.6 to 27.6 mg) was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.4 - 38.5°C

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 26.6 to 27.6 mg

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of replicates:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: relative mean viability

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability compared to the negative control tissues (100%).

Other effects / acceptance of results:

Direct MTT reduction and colour interference:
Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide did not interfere with the MTT endpoint.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 11%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
The standard deviation value of the percentage viability of three tissues treated identically with the reference items was less than 3%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) performed according to OECD439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm²cultured skin ( 26.6 to 27.6 mg). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 11% whereas the test substance showed cell viability of 100%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and the substance does not have to be classified for skin irritation according to Regulation (EC) No. 1272/2008.