1,4-Diazabicyclo[2.2.2]octane-2-methanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 24 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

adopted 26 Jul 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 Jul 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{1,4-diazabicyclo[2.2.2]octan-2-yl}methanol

EC Number:

692-731-2

Cas Number:

76950-43-1

Molecular formula:

C7H14N2O

IUPAC Name:

{1,4-diazabicyclo[2.2.2]octan-2-yl}methanol

Details on test material:

- Name of test material (as cited in study report): [trade name]
- Substance type: off-white powder with lumps
- Analytical purity: 90.7%
- Impurities (identity and concentrations): 1,5-diazabicyclo[3.2.2]nonane-3-ol 8.9%
- Expiration date of the lot/batch: 20 Dec 2014
- Storage condition of test material: at room temperature in the dark in a well-sealed container
- Other: pH: 11.0 at concentration of 10%

Test animals

Species:

human

Strain:

other: EPISKIN-SM™; three-dimensional human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France

TEST METHOD
EPISKIN Small Model™ (EPISKIN-SM™):
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
On the day of receipt, tissues were transferred into 12-well plates and preincubated with prewarmed Maintenance Medium for approx. 26 h at 37 °C.

INCUBATION CONDITIONS
- Temperature (°C): 37 ± 1 (acutal range: 36.6 - 37.5)
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 80 - 100 (actual range: 84 - 88)

Test system

Type of coverage:

open

Preparation of test site:

other: intact human epidermis model

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with PBS served as negative controls, positive controls were exposed to 5% (aq) SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 12.9 to 16.4 mg
The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue.

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: 5% (aq) sodium dodecyl sulphate (SDS) in PBS
The positive control was re-spread after 7 minutes contact time.

Duration of treatment / exposure:

15 min

Observation period:

not applicable

Number of animals:

not applicable
The test was performed in triplicates for each test or control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 15 min
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for approximately 42 h at 37°C.

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, after incubating tissues in maintenance medium for 42 h, tissues were transferred into a 12-wells plate and incubated in 2 mL MTT-medium (0.3 mg/mL) for 3 h at 37 °C. Thereafter the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol. Tubes were stored refrigerated and protected from light for approximately 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. The absorption was corrected for the background absorption.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (PBS). Time point: 15 min. Reversibility: other: not applicable.

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

22

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% (aq) SDS). Time point: 15 min. Reversibility: other: not applicable.

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

93

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 15 min. Reversibility: other: not applicable.

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 93%. Since the mean relative tissue viability for the test substance was above 50% after 15 minutes treatment, the test substance is considered to be non-irritant.

Any other information on results incl. tables

Table 1. Mean absorption in the in vitro skin irritation test 

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570) ± SD	Mean tissue viability (percentage of control)
Negative control	1.315	0.979	1.114	1.136 ± 0.169	100
Test substance	1.006	0.997	1.152	1.052 ± 0.087	93
Positive control	0.173	0.404	0.186	0.254 ± 0.130	22

OD = optical density

SD = standard deviation

Triplicate exposures are indicated by A, B and C.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified
DSD: not classified