Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-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:

The study was conducted between 20 May 2014 and 23 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 439 using a Reconstructed Human Epidermis Test Method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Molecular formula:

C10H14O2

IUPAC Name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Test material form:

other: Liquid

Details on test material:

Identity: IFF TM 09-218
Chemical name: Ethyl bicyclo[2.2.1]hept-5-ene-2 carboxylate
CAS number: 10138-32-6
Intended use: Fragrance ingredient in household products
Appearance: Clear liquid
Storage conditions: Room temperature (ca. 20˚C, in the dark)

Test animals

Species:

other: EpiSkin™ human epidermis skin constructs

Strain:

other: Not applicable

Test system

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: Not applicable

Amount / concentration applied:

10mg/tissue.

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours post-exposure incubation period

Number of animals:

Not applicable

Details on study design:

Reduction of MTT by test substance
It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test substance may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test substance turns
blue/purple after approximately 3 hours incubation at 37 ±2ºC in 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, IFF TM 09-218, was investigated by mixing 10 μL of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of purified water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for colouring potential of test substance
The test substance, IFF TM 09-218, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10 μL of the test substance, with 90 μL of purified water in a transparent container. 100 μL of purified water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was
assessed by eye. If a coloured solution was detected, the tissue staining ability was tested by following the test procedure, however, MTT was replaced with the assay medium and only one tissue for the test substance and the Dulbecco’s phosphate buffered saline (DPBS) control was used.

Receipt of tissues
On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within
the expiry date indicated by the supplier (expiry date: 26 May 2014). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The inserts used for killed tissues were received on a previous occasion (4 February 2014). The tissues had been previously killed by transferring the inserts to a 12 well plate containing 2 mL of water per well and then incubated at 37 ± 2ºC in humidified 5 % CO2 in air for 48 ± 1 hours. The killed tissues were transferred to a 12 well plate after blotting on absorbent
paper and then stored at ca. -20°C until use.

Controls
The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.

The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water.

Preparation/application of samples
The test substance, IFF TM 09-218, a clear liquid, positive and negative controls were in liquid form and were applied by dispensing a volume of 10 µLover each tissue using a positive displacement pipette.

Test procedure
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or
positive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 8 minutes application time. At least one hour before dosing, thawed water killed tissues were transferred into wells of 12
well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 1 hour at room temperature. Triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance (10 ± 2 mg per tissue) at room temperature. As killed tissues may retain somereducing enzymes (which may reduce MTT), triplicate killed tissues were left untreated as a control.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube. When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at room temperature, protected from light, for 4 hours, vortexing after approximately 2 hours.
After formazan extraction, duplicate 200 µL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. Theextractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol
(0.04 N HCl final concentration) as a blank.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean

Value:

23.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes exposure. Remarks: Irritant. (migrated information)

In vivo

Irritant / corrosive response data:

The test substance was predicted to be an irritant. The mean tissue vaibility as a percentage of mean OD negative control for the test substance was 23.9 ± 7.7%.

Any other information on results incl. tables

Possible reduction of MTT by test substance

There was no change in the water control/MTT solution after three hours incubation in the dark at 37 ± 2 °C in a humidified atmosphere of 5% CO2 in air. There was a change in the colour of the test substance, IFF TM 09-218/MTT solution from yellow/green to yellow/green with purple suspension after three hours incubation in the dark at 37 ± 2 °C in a humidified atmosphere of 5% CO2 in air. The test substance had interacted with the MTT, additional controls were used.

Check for colouring potential of test substance

The test substance, IFF TM 09-218/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

Assay validity

Negative control

The mean absorbance of the triplicate negative control values was 0.853 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 7.5 which was below the maximum value of 18.

Positive control

The percentage mean viability of the positive control was 28.1 ± 10.5 of the negative control.These were below the maximum acceptance values of 40% viability and SD of 18%.

EpiSkin™ results

The test substance OD values were corrected for nonspecific MTT reduction by the test substance. The results of the assay are summarised in the table below.

Sample	Tissue viability as percentage of mean OD negative control				Prediction MTT endpoint
	Replicate tissues			Mean±SD
	A	B	c
Negative control	108.1	93.4	98.5	100.0±7.5	Not applicable
Positive control	40.1	23.4	20.7	28.1± 10.5	Irritant
Test substance	18.2	32.6	20.9	23.9± 7.7	Irritant

Nonspecific MTT reduction data

Sample	Tissue replicate	Optical density (OD)	Corrected OD	Corrected OD – Untreated corrected mean OD
			(OD – Blank)
Untreated	A	0.329	0.186	N/A
		0.369	0.226
	B	0.354	0.211
		0.376	0.232
	C	0.361	0.217
		0.365	0.213
	Replicates a, b, c	Mean	0.214
		SD	0.016
Test substance	A	0.289	0.145	-0.042
		0.308	0.165
	B	0.304	0.161
		0.252	0.108
	C	0.371	0.227
		0.368	0.225
	Replicates a, b, c	Mean	0.172
		SD	0.046
Blank		0.138	N/A
		0.148
		0.145
		0.139
		0.146
		0.143
	Mean	0.143
	SD	0.004

sd = Standard Deviation

Note. Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet.

 

EpiSkin™ study data

Sample	Tissue replicate	Optical density (OD)	Corrected OD	Correction for MTT reduction	% negative control
			(OD – Blank)
Negative control	A	1.053	0.910	N/A	108.1
		1.079	0.935
	B	0.925	0.782		93.4
		0.955	0.812
	C	0.974	0.831		98.5
		0.994	0.850
	Replicates a, b, c	Average	0.853		100
		SD	0.059		7.5
Positive control	A	0.509	0.365	N/A	40.1
		0.462	0.318
	B	0.345	0.202		23.4
		0.341	0.197
	C	0.332	0.189		20.7
		0.308	0.165
	Replicates a, b, c	Average	0.239		28.1
		SD	0.082		10.5
Test substance	A	0.259	0.115	0.157	18.2
		0.254	0.111	0.153
	B	0.383	0.240	0.282	32.6
		0.376	0.233	0.275
	C	0.274	0.131	0.173	20.9
		0.286	0.143	0.185
	Replicates a, b, c	Average	0.162	0.204	23.9
		SD	0.059	0.059	7.7
Blank		0.138
		0.148
		0.145
		0.139
		0.146
		0.143
	Average	0.143
	SD	0.004

Correction for MTT reduction = corrected OD – MTT nonspecific reduction value (-0.042)

sd = Standard Deviation

Note. Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that the test substance, IFF TM 09-218, with a mean tissue viability of 23.9 ± 7.7%, was predicted as irritant to the skin.

Executive summary:

The skin irritation potential of the test substance, TM 09-218, was positive according to OECD Test Guideline 439 using a Reconstructed Human Epidermis Test Method. A mean tissue viability of 23.9 ± 7.7%, was predicted and is therefore considered as irritant to the skin.