4-(piperidin-4-yl)morpholine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 - 29 Oct 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

4-(piperidin-4-yl)morpholine

EC Number:

627-580-3

Cas Number:

53617-35-9

Molecular formula:

C9H18N2O

IUPAC Name:

4-(piperidin-4-yl)morpholine

In vitro test system

Test system:

human skin model

Source species:

human

Source strain:

other: EpiDerm™; reconstructed three-dimensional human epidermis (EPI-200)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 23300 (EpiDerm™ Tissues); 102215ZSA (Assay medium); 21675 (EpiDerm™ freeze killed Tissues)
- Delivery date: 27 Oct 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Each tissue was rinsed under a constant soft stream of Dulbecco`s Phosphate-Buffered Saline (DPBS). Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm

NUMBER OF REPLICATE TISSUES: Duplicates of each 2 independent experiments

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes 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%.
- The test substance is considered to be non-corrosive to skin 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%.
The relative mean viability was calculated in the following way:
Relative mean viability (%) = (mean OD562 of the test substance)/(mean OD562 of negative control) x 100

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of the test item

Run / experiment:

3 min

Value:

85.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of the test item

Run / experiment:

60 min

Value:

10.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Yes; an additional procedure using freeze-killed tissues was performed in parallel.
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD values for the negative control are ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: Mean viability of the tissue replicates exposed for 1 hour with the positive control (8N KOH), expressed as % of the negative control, should be < 15%
- Acceptance criteria met for variability between replicate measurements: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%

Any other information on results incl. tables

Table 2: Mean OD₅₆₂ and viabilities for the negative control, positive control and test item

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 min	1.975	1.959	0.023	1.2	100 *
		1.943
	60 min	2.162	2.134	0.040	1.9
		2.106
Positive Control	3 min	0.046	0.076	0.042	N/A	3.9
		0.106
	60 min	0.060	0.056	0.006	N/A	2.6
		0.052
Test Item	3 min	1.724	1.670	0.077	4.6	85.2
		1.615
	60 min	0.186	0.226	0.057	N/A	10.6
		0.266

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

Applicant's summary and conclusion

Interpretation of results:

other: positive according to OECD 431 and according to CLP/EU GHS criteria

Conclusions:

CLP: Skin corrosive Cat 1, H314