Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2009 - 28 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

OECD draft dated 20 March 2009

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1st ATP Council Regulation No 440/2208, Council Regulation No 761/2009 of 23 July 2009

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-cyclohexyl-3-[2-methyl-5-({[(9Z)-nonadec-9-en-1-yl]carbamoyl}amino)phenyl]urea; 3-{2-[({3-[(cyclohexylcarbamoyl)amino]-2-methylphenyl}carbamoyl)amino]cyclohexyl}-1-[5-({[(7Z)-heptadec-7-en-1-yl]carbamoyl}amino)-2-methylphenyl]urea

EC Number:

945-075-9

Molecular formula:

Mixture of C47H74N8O4 and C34H58N4O2

IUPAC Name:

1-cyclohexyl-3-[2-methyl-5-({[(9Z)-nonadec-9-en-1-yl]carbamoyl}amino)phenyl]urea; 3-{2-[({3-[(cyclohexylcarbamoyl)amino]-2-methylphenyl}carbamoyl)amino]cyclohexyl}-1-[5-({[(7Z)-heptadec-7-en-1-yl]carbamoyl}amino)-2-methylphenyl]urea

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: From adult donors; obtained from EpiSkinTM

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): Test No. 1: 09-EKIN-031; Test No. 2: 09-EKIN-045
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 09-EKIN-031 = 15th September 2009; 09-EKIN-045 = 22nd December 2009
- Date of initiation of testing: 15th September 2009 and 22nd December 2009 for test 1 and 2, respectively.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not reported
- Temperature of post-treatment incubation (if applicable): Following 15 minutes test item application and 42 hours post-treatment incubation, the skin sample was placed in MTT solution of 0.5 mg/mL concentration for 3 hours at 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 x 0.5 mL of PBS (PanBiotech GmbH – Batch No 5000209)
- 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: 0.5 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not reported
- Wavelength: 570 nm
- Filter: None reported
- Filter bandwidth: Not applicable

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: In historic negative control data the viability is 86.1 – 113.1 %
- Barrier function: SDS used as a positive control, and the mean viability in historic control data is 2.9 - 23.9 %
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: For both batches of the skin models used the absence of HIV1 and 2, hepatitis C antibodies and the absence of hepatitis B antigen in the donor blood has been confirmed. Additionally, the absence of bacteria, fungus and mycoplasma was verified on the epidermal cells.
- Reproducibility: For the EPISkin batch used in test 1; histology scoring (HES stained vertical paraffin sections. n = 6), specification > 19.5, result was 22.0 ± 0.3 , CV = 1.4 %, and for IC 50 determination (SDS concentration, MTT test, n - 14) specification ≥ 1.5 mg/mL; result was 1.8 mg/mL. For the EPISkin batch used in test 2; histology scoring (HES stained vertical paraffin sections. n = 6), specification > 19.5, result was 22.4 ± 0.4, CV = 1.7 %, and for IC 50 determination (SDS concentration, MTT test, n - 14) specification ≥ 1.5 mg/mL; result was 1.7 mg/mL.

NUMBER OF REPLICATE TISSUES: 3 per test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 3
- Method of calculation used: Optical density

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes of exposure and 42 hours of post-treatment incubation is >50% and the amount IL-1α release is ≤ 60 pg/mL
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): Test No. 1: 09-EKIN-031; Test No. 2: 09-EKIN-045
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 09-EKIN-031 = 15th September 2009; 09-EKIN-045 = 22nd December 2009
- Date of initiation of testing: 15th September 2009 and 22nd December 2009 for test 1 and 2, respectively.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not reported
- Temperature of post-treatment incubation (if applicable): Following 15 minutes test item application and 42 hours post-treatment incubation, the skin sample was placed in MTT solution of 0.5 mg/mL concentration for 3 hours at 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 x 0.5 mL of PBS (PanBiotech GmbH – Batch No 5000209)
- 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: 0.5 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not reported
- Wavelength: 570 nm
- Filter: None reported
- Filter bandwidth: Not applicable

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: In historic negative control data the viability is 86.1 – 113.1 %
- Barrier function: SDS used as a positive control, and the mean viability in historic control data is 2.9 - 23.9 %
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: For both batches of the skin models used the absence of HIV1 and 2, hepatitis C antibodies and the absence of hepatitis B antigen in the donor blood has been confirmed. Additionally, the absence of bacteria, fungus and mycoplasma was verified on the epidermal cells.
- Reproducibility: For the EPISkin batch used in test 1; histology scoring (HES stained vertical paraffin sections. n = 6), specification > 19.5, result was 22.0 ± 0.3 , CV = 1.4 %, and for IC 50 determination (SDS concentration, MTT test, n - 14) specification ≥ 1.5 mg/mL; result was 1.8 mg/mL. For the EPISkin batch used in test 2; histology scoring (HES stained vertical paraffin sections. n = 6), specification > 19.5, result was 22.4 ± 0.4, CV = 1.7 %, and for IC 50 determination (SDS concentration, MTT test, n - 14) specification ≥ 1.5 mg/mL; result was 1.7 mg/mL.

NUMBER OF REPLICATE TISSUES: 3 per test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 3
- Method of calculation used: Optical density

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes of exposure and 42 hours of post-treatment incubation is >50% and the amount IL-1α release is ≤ 60 pg/mL

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

VEHICLE:
- Amount(s) applied (volume or weight with unit): test item moistened with 5 µL distilled water
- Lot/batch no. (if required): Distilled water (ADL PROCHILAB Batch No 100439801)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Distilled water, amount not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
Not reported
- Concentration (if solution): 5 % SDS

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per test; 2 tests

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test 1; Tissue 7

Value:

176.7

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:

Test 1; Tissue 8

Value:

154.7

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:

Test 1; Tissue 9

Value:

59.2

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:

Test 1; mean

Value:

130.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation / corrosion parameter:

other: IL1-α level (pg/mL)

Run / experiment:

Test 1; Mean

Value:

6.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 6

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test 2; Tissue 7

Value:

112.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 7

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test 2; Tissue 8

Value:

98.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 8

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test 2 Tissue 9

Value:

79.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 9

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test 2 Mean

Value:

97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 10

Irritation / corrosion parameter:

other: IL1-α level (pg/mL)

Run / experiment:

Test 2; mean

Value:

3.9

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
- Colour interference with MTT: None reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Negative and positive control values were within the historic ranges

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, mean optical density of the three replicates was ≥ 0.153 (observed 0.250 and 0.384 in tests 1 and 2, respectively).
- Acceptance criteria met for positive control: Yes, mean viability was ≤ 40 % (observed; 12.2 and 10.0 % in tests 1 and 2, respectively.)
- Acceptance criteria met for variability between replicate measurements: Standard Deviation was ≤ 18 for the test item, positive and negative control replicates in Test 2 (observed; 16.3, 1.5 and 8.3, respectively). In test 1, the standard deviation was > 18 for the test item and negative control (observed; 62.5 and 44), therefore, test 2 was conducted to confirm the results obtained with test 1.
- Range of historical values if different from the ones specified in the test guideline: Historic values reported in tables 7 and 8.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin irritation potential of the test item was assessed in guideline human skin model tests and it was concluded that the test item did not meet the requirements for classification.

Executive summary:

The skin irritation potential of the test item was investigated by topical administration on in vitro human reconstructed epidermis in an EU B.46 guideline study. The test item was applied to two Human skin model surfaces (Episkin), during 15 minutes followed by a 42 hours post-incubation period. The study was conducted in triplicate on two occasions with concurrent positive and negative controls. The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT reduction into blue formazan crystal. The concentration of formazan was measured by determining the Optical Density (OD) at 570 nm. The absorbance was measured in triplicate of MTT extract. The measured absorbances are proportional to the number of live cells. Additionally, to confirm the results of the tests, the released IL1-α by the epidermis in the assay medium was measured (on frozen medium) using an ELISA test.

In test 1, the mean viability of the epidermis skins was 130.2% (considered as 100%), versus 12.2% in the positive control (5% SDS). To confirm these results, the IL1-α released by the epidermis was measured; the mean concentration measured was 6.8 pg/mL, versus 157.9 pg/mL in the positive control and 7.5 pg/mL in the negative control. Due to the high variability in the optical density obtained in the three epidermis treated in a same way in Test 1, a second experiment was conducted to confirm the non classification of the test item. The mean viability of the epidermis skins was 97.0%, versus 10.0% in the positive control. The mean concentration of ILl-α released was 3.9 pg/mL, versus 77.7 pg/mL in the positive control and 10.2 pg/mL in the negative control. Based on these results, the test item is not classified for skin irritation under UN GHS regulations.

The study is a GLP compliant guideline experiment study acceptable without restrictions for assessment of this endpoint.