N-cyclohexyl-N-methylcyclohexylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31/05/2017-02/06/2017

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:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: July 28th, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclohexyl-N-methylcyclohexylamine

EC Number:

231-453-4

EC Name:

N-cyclohexyl-N-methylcyclohexylamine

Cas Number:

7560-83-0

Molecular formula:

C13H25N

IUPAC Name:

N-cyclohexyl-N-methylcyclohexanamine

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MDCHA KD-1050649
- Expiration date of the lot/batch: January 13, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The sample of test substance has been stored in the delivered package, in dark at room temperature during the study.
- Stability under test conditions: January 13, 2018

In vitro test system

Test system:

human skin model

Remarks:

a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: the tissue for research purposes from accredited institutions

Source strain:

other: Keranocyte strain: 00267

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SK)
- Tissue batch number(s): Lot No. 23815, kit C
- Certificate of Analysis date: 31.5.2017
- Date of initiation of testing: 31.5.2017


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance (controls)
Detailed procedure is described in internal SOP M/46/3.
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg·mL-1
- Incubation time: 3 hr
- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed (frozen) tissues
- N. of replicates : 2
- Method of calculation used:
Data correction for direct MTT interference
True viability = % Viability of treated tissue – % NSMTT
% NSMTT= TS OD570 (frozen tissues)/NC OD570 (live tissues)*100%
The net OD of the test substance treated killed control was subtracted from the mean OD of the test substance treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction - functional check in tubes
2. Direct MTT reduction – test in frozen tissues
3. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) 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 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Amount/concentration applied:

test substance C1: 50 μL of the test substance
NC: 50 μL water tested with every exposure time
PC: 50 μL 8N KOH tested with every exposure time

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min

Run / experiment:

1

Value:

108.8

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 min

Run / experiment:

2

Value:

6.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:

- Direct MTT reduction - functional check in tubes
The test substance reduces MTT directly.

- Direct-MTT reduction:
Average TS OD570 after 3 minutes was lower than control OD570 so no correction was performed after 3 minutes treatment.
Average TS OD570 after 60 minutes was a bit higher than average OD570 of the NC. The difference was 0.075 what is 4.4% of viability of live negative control. OD570 from the MTT test was corrected by subtraction of the 4.4% of viability.

- Colour interference with MTT:
Colour interference was not assayed, because the test substance is colourless. Slight blue colouring occurred at rinsing in rests after rinsing, but no colour was perceptible in treated tissues after rinsing.

ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.

Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.677 (3 min) and 1.691 (60 min) which is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 5.8 % which is ≤15%.
Coefficient of variance: CV values in all triplets of tissues were ≤ 0.3 in all cases

Any other information on results incl. tables

Direct MTT reduction: test in frozen tissues

time	label	treatment	OD570			SD	CV	difference
(min)			Tissue 1	Tissue 2	avg	[%]
3	NC	water	0.070	0.069	0.069	0.000	0.000
	C1	87/17	0.066	0.058	0.062	0.004	0.065	-0.007
60	NC	water	0.063	0.067	0.065	0.002	0.031
	C1	87/17	0.142	0.137	0.140	0.002	0.018	0.075

Average TS OD₅₇₀after 3 minutes was lower than control OD₅₇₀so no correction was performed after 3 minutes treatment.

Average TS OD₅₇₀after 60 minutes was a bit higher than average OD₅₇₀of the NC. The difference was 0.075 what is 4.4% of viability of live negative control. OD₅₇₀from the MTT test was corrected by subtraction of the 4.4% of viability.

MTT test

time	treatment		OD570						% NC
			tissues			mean	SD	CV
			1	2	3
3min	NC	water	1.759	1.781	1.490	1.677	0.132	0.079	100.0
	C1	87/17	2.014	1.679	1.778	1.824	0.141	0.077	108.8
	PC	8N KOH	0.125	0.129	0.113	0.122	0.007	0.056	7.3
	NC	water	1.614	1.745	1.715	1.691	0.056	0.033	100.0
60 min	C1	87/17	0.206	0.178	0.168	0.184	0.016	0.087	10.9
	PC	8N KOH	0.085	0.100	0.108	0.098	0.010	0.098	5.8

Value of OD₅₇₀in the extract from the first tissue in 3 min treatment was rather higher. At the same time visibly lower volume was observed in the well.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the above-described experimental design, the test substance N-methyldicyclohexylamine was corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

The test substance N-methyldicyclohexylamine was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: July 28th, 2015.

Interference of colour with the endpoint was not observed. The test substance did not change colour during the test.

Test for direct reduction in test tubes was performed simultaneously. The test substance changed colour of MTT medium to purple. The test substance is directly reducing. For correction of result of MTT test, the test in frozen tissues was performed for determination of part of reduction of MTT caused directly by the test substance.

In the MTT test, the test substance (50 μL) was placed atop the tissue and spread onto all tissue surface. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C1), three for the positive control (PC) and three for the negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol for two hours at room temperature with shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, the average viability of tissues treated with the test substance was 108.8 % of the negative control average value after 3 minutes treatment which is ≥50% and 6.4 % after 60 minutes treatment which is ≤15%.

This part of evaluation according to OECD TG 431 is valid for the obtained results, because of prediction criterium:

“The test substance is considered to be corrosive to skin:

ii) 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 %.”

The test substance N-methyldicyclohexylamine was corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.