Reaction mass of 1,5-naphthylene diisocyanate and Amines, hydrogenated tallow alkyl

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 November 2017 to 14 November 2017

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:

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:

updated 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Molecular formula:

C24H32N4O2

IUPAC Name:

Reaction product of 1,5-naphthylene diisocyanate (221-641-4) and cyclohexylamine (203-629-0)

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human-derived

Justification for test system used:

Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s):
17-EKIN-045
- Production date:
Not reported
- Shipping date: Not reported
- Delivery date: Not reported
- Date of initiation of testing:
Pre-incubation started on day of tissue arrival. Tissues were transferred to maintenance medium and incubated at 37 °C, 5% CO2 in air overnight.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
- Temperature of post-treatment incubation (if applicable):
37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP:
None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
2 mL of 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer:
Labtech LT-4500 microplate reader
- Wavelength:
570 nm
- Filter: None
- Filter bandwidth: Not applicable

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results. The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

An assessment of colour interference with the MTT endpoint was also performed. 10 mg of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made. The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours incubation is less than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2)

VEHICLE : Not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): As supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

89.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

96.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

97.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

94.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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
- Range of historical values if different from the ones specified in the test guideline: Not reported

Any other information on results incl. tables

Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.786	0.789	0.061	99.6	100*	7.8
	0.730			92.5
	0.852			108.0
Positive Control Item	0.212	0.243	0.027	26.9	30.8	3.4
	0.262			33.2
	0.524			32.2
Test item	0.708	0.746	0.033	89.7	94.5	4.2
	0.759			96.2
	0.770			97.6

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean viability of test item treated tissues after a 15 minute exposure period followed by 42 hour post-exposure period was 94.5 %. The test item was classified as non-irritant under the EU CLP and UN GHS classification criteria.

Executive summary:

The skin irritancy potential of the test item was assessed in an in vitro EPISKIN Reconstructed Human Epidermis Model test according to OECD 439 and EU B.46 test guidelines. Dulbecco's Phosphate Buffer (DPBS) with Ca2 + and Mg2 + and 5 % w/v Sodium dodecyl sulphate (SDS) were included as negative and positive controls, respectively. The relative mean viability of the test item treated tissues was 94.5 % after a 15 minute exposure period followed by 42 hour post-exposure period. It was considered unnecessary to perform IL-1 analysis as the MTT test was unequivocal. The test item was classified as non-irritant under the EU CLP and UN GHS classification criteria.

The study is a GLP compliant, guideline experimental study with no restrictions and therefore fully adequate for assessment of this endpoint.