Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 February 2016 to 22 February 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

EC Number:

267-032-7

EC Name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Cas Number:

67762-73-6

IUPAC Name:

Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): EXP1506833
- Physical state: amber coloured viscous liquid
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: E00350-290
- Expiration date of the lot/batch: 23 June 2019
- Purity test date: 58 %

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

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN reconstructed human epidermis

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 16 February 2016
EpiSkinTM Tissues (0.38cm²) lot number: 16-EKIN-007
Maintenance Medium lot number: 16-MAIN3-009
Assay Medium lot number: 16-ESSC-007

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium Dodecyl Sulphate
- Concentration (if solution): 5 % w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean

Value:

110.9

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:

1

Value:

113

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

106.4

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

113.3

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless, it was therefore unnecessary to run color correction tissues.

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

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	±SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	±SD of relative mean viability
Negative Control Item	0.865	0.845	0.024	102.3	100*	2.8
	0.819			96.9
	0.852			100.8
Positive Control Item	0.063	0.071	0.012	7.5	8.4	1.3
	0.065			7.7
	0.084			9.9
Test Item	0.955	0.937	0.033	113.0	110.9	3.9
	0.899			106.4
	0.957			113.3

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability calculated as a percentage of the negative control was 110.9 % after the 15-minute exposure followed by the 42-hour post exposure. The test substance did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vitro skin irritation of the test substance was determined in accordance with the OECD Testing Guideline 439. The purpose of this test was to evaluate the skin irritation potential of the registered substance using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the registered substance treated tissues relative to the negative controls.

Triplicate tissues were treated with the registered substance for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean tissue viability calculated as a percentage of the negative control was 110.9 % after the 15-minute exposure followed by the 42-hour post-exposure. The test substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.