Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-08-11 to 2021-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT), For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM

Version / remarks:

2014-11-07

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008, Annex Part B, B.40bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, amended by Commission Regulation (EU) 2019/1390 of 31 July 2019.

Version / remarks:

2019-07-31

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

EC Number:

954-590-8

Cas Number:

2522560-40-1

Molecular formula:

C31H56N4O2

IUPAC Name:

(1E)-N-[4-({4-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]cyclohexyl}methyl)cyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

other: human adult donors

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This method is approved by international regulatory agencies as a replacement for the identification of corrosives in the in vivo Rabbit skin assay (OECD 404) and is specifically approved as a replacement for the in vivo skin corrosivity test within OECD 431.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)
- Tissue batch number: 34188
- Expiry date: 2021-08-20
- Date of initiation of testing: 2021-08-18

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (3 min treatment), 37±1 °C, (1 hour treatment)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The units were rinsed thoroughly with 1x DPBS sterile solution to remove all of the test material from the epidermal surface. The constant soft stream of DPBS was used from approximately 1.5 cm distance, and filling and emptying the tissue insert was completed 20 times.The rest of the DPBS was decanted onto the absorbent material.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL of 1 mg/mL MTT per well
- Incubation time: 3 hours (± 15 min)
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability, barrier function and reproducibility: The quality of the final product is assessed via the MTT cell viability test using the cytotoxic test compound Triton X-100 (1.0%)
- Contamination: The absence of HIV1 and 2 antibodies, Hepatitis C antibodies and Hepatitis B antigens HBs as well as funghi, bacteria and mycroplasma were verified beforehand.

NUMBER OF REPLICATE TISSUES: 3 replicate tissues per test item and negative/positive control (for each exposure time)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues: The test item did not show direct interaction with MTT. Using of additional fresh tissue controls was not necessary.
- Killed tissues: The test item did not show direct interaction with MTT. The use of additional killed tissue controls was not necessary.
- Method of calculation used: See "Any other information on materials and methods, incl. tables"

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 min exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%. If a substance identified as corrosive shows a viability of less than 25% after 3 min exposure, it can be assigned to sub-category 1A. If a substance identified as corrosive shows a viability of equal or higher than 25% after 3 min exposure, it can be assigned to a combination of sub-categories 1B and 1C.
- The test substance is considered to be non-corrosive to skin if the viability after 3 min exposure is higher or equal to 50% or if the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: Approx. 50 μL

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration: 8N KOH solution

Duration of treatment / exposure:

3 min and 1 hour

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

3 replicate tissues per test item, negative control and positive control (for each exposure time) were tested in a single independent experiment.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure (mean of 3 tissue replicates)

Value:

84

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:

60 min exposure (mean of 3 tissue replicates)

Value:

70

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: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study (Study No.: 392-431-5488) using the ten Proficiency Chemicals according to OECD Test Guideline No. 431.

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: See "Attached background material"

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

This test method (OECD 431) must be considered together with a suitable test method for identifying skin irritants. The final classification is chosen based on the results from both test guidelines will be described in the endpoint summary.

Conclusions:

In this in vitro skin corrosion test in EpiDerm model according to OECD 431 and GLP with the test item, the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.

Executive summary:

A GLP-compliant EpiDerm test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, 18 June 2019. Disks of EpiDermTM (three units / chemical / incubation time) were treated with the test item and incubated for 60 minutes (+ 3 min) at standard culture condition (37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere) and 3 min at room temperature. Exposure of test material was terminated by rinsing with DPBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±5 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using MTT-100-EXT (isopropanol) and quantified spectrophotometrically. Potassium hydroxide (KOH) 8N solution and sterile deionized water treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 60 minutes of exposure is above or equal 15 % and the mean relative viability after 3 minutes of exposure is above or equal 50 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 60 minutes and 3 minutes of exposure. The average test item treated tissue mean relative viability was 70 % at 60 minutes of exposure and the mean relative viability was 84 % at 3 minutes of exposure. Positive and negative controls showed the expected optical density (OD) and cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid. In conclusion, in this in vitro skin corrosion test in EpiDermTM model (OECD 431), the results indicate that the test item is not corrosive. According to the UN GHS classification systems, the test item has been categorized as “Non-corrosive”.