Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August - 15 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane

EC Number:

207-428-9

EC Name:

1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane

Cas Number:

470-67-7

Molecular formula:

C10H18O

IUPAC Name:

1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane

Constituent 2

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

impurity 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

impurity 2

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

impurity 3

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

impurity 4

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 5

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 6

Reference substance name:

(+)-pin-2(3)-ene

EC Number:

232-087-8

EC Name:

(+)-pin-2(3)-ene

Cas Number:

7785-70-8

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 7

Reference substance name:

(-)-pin-2(3)-ene

EC Number:

232-077-3

EC Name:

(-)-pin-2(3)-ene

Cas Number:

7785-26-4

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 8

Reference substance name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

Cas Number:

116724-26-6

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

impurity 9

Reference substance name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

Cas Number:

7378-37-2

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

impurity 10

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 11

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 12

Reference substance name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

EC Number:

208-083-7

EC Name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

Cas Number:

508-32-7

Molecular formula:

C10H16

IUPAC Name:

1,7,7-trimethyltricyclo[2.2.1.0~2,6~]heptane

impurity 13

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 14

Reference substance name:

3-isopropyl-(6S)-methylenecyclohexene

Cas Number:

6153-16-8

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6S)-methylenecyclohexene

impurity 15

Reference substance name:

3-isopropyl-(6R)-methylenecyclohexene

Cas Number:

6153-17-9

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6R)-methylenecyclohexene

impurity 16

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,4-diene

impurity 17

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methyleneocta-1,6-diene

impurity 18

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 179011
Purity: 76.9% (sum of the two main constituents)
Name of test material (as cited in study report): REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 03 April 2017

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 16-RHE-098) were received on 13 September 2016.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 16 MPE 094) during 2 hours and 5 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch No. 16 MA 051).

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS – PAN BIOTECH GmbH - Batch No. 4710416) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask (QS 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 4710416). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 41-hour and 41-minute post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measured OD were proportional to the number of living cells. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1. The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%. In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41-hour and 41-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 replicates of living human skin models

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
No blue or purple coloration was noted after 3 hours of incubation between 37.4°C and 37.8°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colorless solution was obtained in water after 3 hours of incubation between 37.4°C and 37.8°C, 5% CO2. A colorless solution was obtained in isopropanol after 2 hours of incubation at room temperature. No significant coloration appeared. Therefore the test item was considered to be not interfering with the MTT assay.


MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 1.4%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate).

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.829	0.885	1.053	84.0	100.0	14.2
		0.912
		0.916
	2	1.059	1.102		104.7
		1.127
		1.121
	3	1.146	1.172		111.3
		1.165
		1.205
Positive control	1	0.012	0.012	0.015	1.1	1.4	0.2
		0.012
		0.013
	2	0.015	0.015		1.4
		0.016
		0.016
	3	0.017	0.017		1.6
		0.017
		0.017
Test item	1	0.014	0.014	0.014	1.3	1.4	0.1
		0.015
		0.015
	2	0.014	0.015		1.4
		0.015
		0.016
	3	0.014	0.014		1.3
		0.014
		0.015

#: mean of 3 values (triplicate of the same extract)

OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria: SD ≤ 18%

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. It is also classified in Category 2 according to UN GHS regulation.
Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or in Category 2 “Irritant”.

Executive summary:

An in vitro skin irritation test using the Reconstructed human Epidermis (SkinEthic RHE® model) was performed according to OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test substance.

The test substance was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 41-minute post-incubation period at 37°C, 5% CO₂. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean corrected percent viability of the treated tissues was 1.4%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Therefore, in accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. It is also classified in Category 2 according to UN GHS regulation.

Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or in Category 2 “Irritant”.