[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

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:

2016-02-25 to 2016-09-22

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:

2015-06-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

EC Number:

260-694-8

EC Name:

[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

Cas Number:

57357-85-4

Molecular formula:

C11H21N

IUPAC Name:

1-[(1S,2S,3S,5R)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl]methanamine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: WE 20PA15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS:
- Liquid / colorless, clear
- pH ~7, undiluted test substance

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: 00267

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm EPI-200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23317

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min exposure at room temperature, 1 h exposure at 37 °C.
- Temperature of post-treatment incubation: 3 h at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: at least one washing step with PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.567 ± 0.068 (MTT QC assay, 4 h, n = 3; acceptance criteria: OD(540 - 570 nm) [1.0 - 3.0])
- Barrier function: 6.7 h (ET-50 assay, 100 µL 1 % tritinX-100, 4 time points, n = 3, MTT Assay; acceptance criteria: ET-50 [4.77 -8.72 h])
- Contamination: No


NUMBER OF REPLICATE TISSUES:
2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed EPI-200 tissues
- Procedure used to prepare the killed tissues: killed by freezing at -20 °C
- No. of replicates : 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50 %. In addition, those materials with a viability of > 50 % after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min, 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

13.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: yes

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

Any other information on results incl. tables

Table 1: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation.

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	1.550	1.836	1.693
		Viability [% of NC]	91.6	108.4	100	11.9	11.9
	KC tissues	Mean OD570	0.115			0.115
		Viability [% of NC]	6.8			6.8
Test substance	Viable tissues	Mean OD570	0.607	0.771	0.689
		Viability [% of NC]	35.9	45.5	40.7	6.8	16.8
	KC tissues	Mean OD570	0.695	0.054	0.695
		Viability [% of NC]	41.0	3.2	41.0
	Final mean viability of tissues after KC correction [% of NC]***:				0.0
PC	Viable tissues	Mean OD570	0.330	0.332	0.331
		Viability [% of NC]	19.5	19.6	19.5	0.1	0.5

* due to organizational reasons, only 1 KC-tissue was used for the NC

** the value of KC-tissue 2 is attributed to be an outlier, therefore it was not used for calculation

*** negative value is set to zero

Table 2: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values,

standard deviations and coefficient of variation

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	1.701	1.716	1.708
		Viability [% of NC]	99.6	100.4	100.0	0.6	0.6
	KC tissues	Mean OD570	0.090	0.107	0.098
		Viability [% of NC]	5.3	6.2	5.8	0.7	11.9
Test substance	Viable tissues	Mean OD570	0.777	1.017	0.897
		Viability [% of NC]	45.5	59.5	52.5	9.9	18.9
	KC tissues	Mean OD570	0.628	0.718	0.673
		Viability [% of NC]	36.8	42.0	39.4	3.7	9.4
	Final mean viability of tissues after KC correction [% of NC]***:
PC	Viable tissues	Mean OD570	0.096	0.085	0.090
		Viability [% of NC]	5.6	4.9	5.3	0.5	8.6

Applicant's summary and conclusion

Interpretation of results:

Category 1 (corrosive) based on GHS criteria