Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2016 - 22 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only.

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)

Version / remarks:

Adopted 28 July 2015

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:

Based on a "Statement on the Scientific Validity of In Vitro Tests for Skin Irritation" (November 2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

3rd revision (2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

EC Number:

273-453-7

EC Name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Cas Number:

68966-86-9

Molecular formula:

C14H22O2

IUPAC Name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

other:

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Human donation
- Tissue: normal epidermal keratinocytes

Justification for test system used:

Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. Skin irritation is defined in Section 3.2.1.1 of Annex I to the CLP regulation as “...the production of reversible damage of the skin following the application of a test substance for up to 4 hours”. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT Kit
- Tissue batch number(s): 23314
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: 16 February 2016
- Date of initiation of testing: Pre-incubation phase started 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 ºC
- Temperature of post-treatment incubation (if applicable): 37 ± 1 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 300 μL, one washing step
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Not applicable, microplate reader used
- Wavelength: 570 ± 1 nm
- Filter: Not reported
- Filter bandwidth: 570 ± 1 nm
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control; 4.77 %
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: None reported
- Reproducibility: Relative standard deviation for mean viability; 14.79 % postive control. Relative standard deviation for mean absorption; 16.6 % postive control, 8.55 % negative control.

NUMBER OF REPLICATE TISSUES: 3 per control and test item treatment

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 43.5 hours exposure is less than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL
- Concentration (if solution): Undiluted test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): Not reported

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5 % SLS in deioinsied water; concentration not reported

Duration of treatment / exposure:

43.5 hours

Duration of post-treatment incubation (if applicable):

67.5 hours

Number of replicates:

3 per test item, negative or positive control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: mean tissue absorbance

Remarks:

Of three wells after blank corrction

Run / experiment:

1

Value:

1.615

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Of three wells after blank

Run / experiment:

2

Value:

1.338

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Of three wells after blank correction

Run / experiment:

3

Value:

1.495

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

other: Mean tissue absorbance

Remarks:

Mean of three tissue after blank correction

Run / experiment:

Mean

Value:

1.483

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

TIssue

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

Tissue 2

Value:

85.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation / corrosion parameter:

other: Relative absorbance (%)

Remarks:

compared to blank

Run / experiment:

Tissue 3

Value:

95.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation / corrosion parameter:

other: Mean relative abosrbance

Remarks:

% of negative control

Run / experiment:

Mean

Value:

94.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Did not show blue colour after 1 hour incubation
- Colour interference with MTT: No colour change

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, within the required range of OD ≥ 0.8 and ≤ 2.8
- Acceptance criteria met for positive control: Yes, induced a decrease in absorbance of 5.1 % compared to the negative control
- Acceptance criteria met for variability between replicate measurements: Not reported
- Range of historical values if different from the ones specified in the test guideline:Not applicable

Any other information on results incl. tables

Results of treatment with test item and the controls

Dose Group	Exposure Interval	Tissue No.	Absorbance 570 nm (Well 1)	Absorbance 570 nm (Well 2)	Absorbance 570 nm (Well 3)	Mean Absorbance of 3 Wells*	Mean Absorbance of three wells blank corrected	Mean Absorbance of three tissues after blank correction	Rel. Absorbance (%) 1, 2 + 3**	Relative Standard Deviation (%)	Mean. Rel. Absorbance (% of negative control)***
Blank			0.037	0.038	0.038	0.038	0.000
Negative Control	60 mins	1	1.540	1.548	1.508	1.532	1.495	1.563	95.6	4.1	100.0
		2	1.644	1.591	1.593	1.609	1.572		100.6
		3	1.717	1.631	1.630	1.659	1.622		103.8
Positive Control	60 mins	1	0.117	0.120	0.118	0.118	0.081	0.080	5.2	4.4	5.1
		2	0.118	0.107	0.116	0.113	0.076		4.8
		3	0.104	0.132	0.124	0.120	0.083		5.3
Test item	60 mins	1	1.672	1.634	1.652	1.653	1.615	1.483	103.4	9.4	94.9
		2	1.384	1.362	1.380	1.375	1.338		85.6
		3	1.530	1.528	1.540	1.532	1.495		95.7

 * Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 100*(absorbance_tissue)/ (mean absorbance_{negative control})

***relative absorbance per treatment group (rounded values): 100*(mean absorbance_{test item/positive control})/ (mean absorbance_{negative control})

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was non-cytotoxic in an experiment employing an artificial three-dimensional model of human skin. Conducted according to OECD 439 (EU B.46) and GLP, the in vitro study is considered reliable without restriction (Klimisch 1). The test item should not be classified as an irritant (UN GHS no category).

Executive summary:

Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt. Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not significantly decrease (94.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.   The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).