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

Endpoint summary

Description of key information

The skin corrosivity of the test substance, TM 09-218, was determined according to OECD Test Guideline 431 using Reconstructed Human Epidermis (RHE) Test Method. IFF TM 09-218, elicited a mean tissue viability of 94.7% for three minute contact and 80.5% for one hour contact and was predicted as non-corrosive.
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.
The eye irritation potential of the test substance, TM 09-218, was assessed as not irritating according to OECD Test Guideline 437 using the Bovine Corneal Opacity & Permeability Assay method.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

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)

Species:

other: EpiSkin™ human epidermis skin constructs

Strain:

other: Not applicable

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.

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)

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%.

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 June 2014 and 11 June 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 437 using the Bovine Corneal Opacity & Permeability Assay method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Eyes from adult cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes
The bovine eyes, supplied by a reputable supplier, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 12:00 hours, 10 June 2014). Eyes were obtained from cattle aged approximately 25 months. Instructions were given to avoid damaging the corneas
during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v)
penicillin/streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of excision (incubation of mounted corneas commenced at 14:48 hours, 10 June 2014).

Vehicle:

other: 0.9% Sodium chloride solution

Controls:

yes

Amount / concentration applied:

750 µL of the test substance.

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Observation period (in vivo):

Two hours ± 10 minutes

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Preparation of corneas
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been dissected.
Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting.
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently
flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% penicillin/streptomycin, using a syringe. The posterior
compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20°C).
After overnight incubation at room temperature (approximately 20°C) the HBSS plus 1% penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. The corneas were then incubated in the upright position for 60 minutes ± 5 minutes at 32°C ± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout
the experiment.
At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM (posterior chamber first). Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Opacity measurement
The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.

Test substance pH
An estimate of the pH of the test substance, diluted 1 in 10 with 0.9% saline, was determined using pH test sticks and recorded.

Treatment groups
Corneas were treated in triplicate with either the test substance, positive control (ethanol) or negative control (0.9% sodium chloride solution).

Treatment of corneas
The test substance and the positive control were tested undiluted. Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty µL (750 µL) of the test substance, positive control or negative control was introduced into the anterior part of each holder. Following application, the anterior compartment was plugged and the holder turned to a horizontal position and slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. The test substance or controls were in contact with the cornea for a total of 10 minutes (± 30 seconds). Each holder was incubated in a horizontal position at 32°C ± 1°C in a waterbath.
Following incubation, the test substance, positive and negative controls were removed from the epithelial surface of the cornea by washing, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on
the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The test substance required
three washes.
The anterior compartment was then filled with cMEM taking care to ensure no air bubbles were present in the compartment. Once all the air bubbles
had been removed, the anterior compartment was re-plugged and the holders returned to the waterbath and incubated, in an upright position, for two hours ± 10 minutes at 32°C ± 1°C.
Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score. Throughout the assay the corneas were examined for opaque spots or other irregularities.

Permeability determination
The sodium fluorescein stock solution (4 mg/mL) was thawed prior to commencing the permeability incubation. Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One mL (1 mL) of the sodium fluorescein solution was added to the anterior compartment using a micropipette. Following addition of the sodium fluorescein solution to the anterior side of the holder, the compartment was
plugged and the corneas incubated in a horizontal position at 32°C ± 1°C for 90 ± 5 minutes in a waterbath.
Following incubation, the medium in the posterior compartment was mixed by drawing approximately 2.5 mL gently up and down a 5 mL syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 490 nm (OD490) and a sample of cMEM read (OD = 0.064). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Irritation parameter:

other: In vitro irritancy score

Basis:

mean

Time point:

other: 2 hours

Score:

2.8

Reversibility:

not specified

Irritant / corrosive response data:

Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with test substance, IFF TM 09-218, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas treated with the negative control, 0.9% sodium chloride solution, were clear.

Sample: IFF TM 09-218

0.9 % sodium chloride solution (negative control)

Cornea	Opacity pre-	Opacity post-	Change	Permeability OD 490
1	6	8	2	0.019
2	5	6	1	0.014
5	4	5	1	0.016
Mean change			1.333	Mean 0.016
SD			0.577	SD 0.003

 

Sample: IFF TM 09-218

Cornea	Opacity pre-	Opacity post-	Change	Corrected change	Permeability OD 490	Corrected
9	6	11	5	3.667	0.040	0024
10	4	7	3	1.667	0.020	0.004
11	6	10	4	2.667	0.015	-0.001
Mean change				2.667	Mean corrected OD490	0.009
SD				1.000	SD	0.013

 

In vitro irritancy score of IFF TM 09-218

(Opacity + (15 x corrected OD490))

Cornea	Opacity	15 x OD490	In vitro irritancy score
9	3.667	0.355	4.022
10	1.667	0.055	1.722
11	2.667	-0.020	2.647
Mean in vitro irritancy score			2.8
SD			1.2

SD = Standard deviation

Note. Rounded values only are displayed, unrounded numbers are used for calculations by excel spreadsheet.

 

Sample: Ethanol (positive control)

0.9 % sodium chloride solution (negative control)

Cornea	Opacity pre-	Opacity post-	Change	Permeability OD 490
1	6	8	2	0.019
2	5	6	1	0.014
5	4	5	1	0.016
Mean change			1.333	0.016
SD			0.577	0.003

 

Sample: Ethanol (positive control)

 

Cornea	Opacity pre-	Opacity post-	Change	Corrected change	Permeability OD 490	Corrected
3	6	36	30	28.667	0.930	0.914
6	4	28	24	22.667	0.995	0.979
8	6	36	30	28.667	1.180	1.164
Mean corrected change				26.667	Mean corrected OD 490	1.019
SD				3.464	SD	0.130

 

In vitro irritancy score of ethanol (positive control)

(Opacity + (15 x corrected OD490))

Cornea	Opacity	15 x OD490	In vitro irritancy score
3	28.667	13.705	42.372
6	22.667	14.680	37.347
8	28.667	17.455	46.122
Mean in vitro irritancy score			41.9
SD			4.4

 

SD = Standard deviation

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

Assay validity

The positive control, ethanol, elicited an In Vitro Irritancy Score of 41.9. This value was within the historical range (mean ± 2 x SD = 25.0 – 49.8) for the assays performed to date. The negative control, 0.9% sodium chloride solution, opacity mean change value was 1.333 which was below the maximum acceptance value of 2.0. The permeability mean of the negative control was 0.016, which was below the maximum acceptance value of 0.1.

pH determination

The pH of the test substance, IFF TM 09-218, diluted 1 in 10 in 0.9% saline, measured using pH sticks, was approximately 7.0.

The results of the BCOP assay are summarised in the table below.

Sample	Opacity±SD	Permeability±SD	In vitro irritancy score±SD	In vitro classification
Test substance	2.667 ± 1.000	0.009 ± 0.013	2.8 ± 1.2	No category
Ethanol	26.667 ± 3.464	1.019 ± 0.130	41.9 ± 4.4	No prediction can be made
0.9 % sodium chloride solution	1.333 ± 0.577	0.016 ± 0.003	Not applicable	Not applicable

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that IFF TM 09-218 was predicted to have a classification of No Category for ocular irritation according to the UN GHS.

Executive summary:

The eye irritation potential of the test substance, TM 09-218, was assessed as not irritating according to OECD Test Guideline 437 using the Bovine Corneal Opacity & Permeability Assay method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion / irritation

Skin corrosion is defined as the production of irreversible damage to the skin, namely visible necrosis through the epidermis and into the dermis following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of observations at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars. Skin irritation is defined as the production of reversible damage to the skin following the application of a test substance for up to 4 hours. Several factors are considered in determining the corrosion and irritation potential of substances before in vivo testing is undertaken.

The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. To this end, an in vitro study was performed to assess the potential for skin corrosion using the EpiDerm^TM 3D human skin Model.

The in vitro EpiDerm^TMskin corrosion test (EPI-200-SCT) using reconstructed human epidermis, supplied by MatTek Corporation, was accepted as a replacement for the in vivo skin corrosion test by the European Centre for the Validation of Alternative Methods (ECVAM) on 20 March 2000.

The test involves the application of the test substance for 3 minutes and for 1 hour to the EpiDerm™ three-dimensional human skin model. The model consists of normal, human derived epidermal keratinocytes which have been cultured on 0.6 cm² inserts to form a multi-layered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum. The cultured tissues were supplied as kits by MatTek Corporation, Ashland, Massachusetts, USA. The kits included assay medium, plates and the tissues, which were shipped on agarose in sealed pouches.

The principle of the assay is that corrosive materials are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) at 3 minute and 1 hour exposure times to identify corrosive and non-corrosive substances. The test includes acceptance criteria for both negative and positive controls.

The test substance, TM 09-218, elicited a mean tissue viability of 94.7% for three minute contact and 80.5 % for one hour contact and was predicted as non-corrosive in the EpiDerm™ skin corrosivity test.

 

An in vitro skin irritation test was performed on the test substance, TM 09 -218. The EpiSkin™ Skin Irritation Test using reconstructed human epidermis skin constructs.

The EpiSkin™ Skin Irritation Test (15 min - 42 hours) using reconstructed human epidermis skin constructs supplied by SkinEthic Laboratories, Lyon, France, was accepted as a replacement to the in vivo Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods (ECVAM) in a statement dated 27 April 2007.

The test involves the application of the test substance for 15 minutes to the EpiSkin™ three dimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multi-layered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38 cm².

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell damage in the cell layers. The cell viability is determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3 (4,5 dimethylthiazol 2 yl) 2, 5 diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

The test substance, IFF TM 09-218, elicited a mean tissue viability of 23.9 ± 7.7 % and was predicted as irritant to the skin.

 

Eye irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay of vision following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

Eye irritation means the production of changes in the eye following application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application. The classification system for substances involves a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage. Substances that have the potential to seriously damage the eyes are classified in Category 1 as they have irreversible effects. Substances that have the potential to induce reversible eye irritation are classified in Category 2 as irritating to the eyes.

The eye irritation potential of the test substance was assessed according to the Bovine Corneal Opacity & Permeability Assay (BCOP) study. This was developed by Pierre Gautheron et al. (1992) as an in vitro alternative to the in vivo Draize Eye Irritation test. It is an organotypic model that uses isolated bovine corneas from freshly slaughtered cattle as a means of assessing the potential of a test substance to cause serious eye damage. Two endpoints, corneal opacity and permeability, are measured and combined to give an In Vitro Irritancy Score which is used to assign an in vitro irritancy hazard classification category for prediction of the ocular irritation potential of a test substance.

The BCOP test method was evaluated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), in conjunction with the European Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM), in 2006 and 2010. In the first evaluation (ICCVAM 2006), the BCOP test method was evaluated for its usefulness to identify chemicals (substances and mixtures) inducing serious eye damage. In the second evaluation (ICCVAM 2010), the BCOP test method was evaluated for its usefulness to identify chemicals (substances and mixtures) not classified for eye irritation or serious eye damage.

From these evaluations and their peer review it was concluded that the test method can correctly identify chemicals (both substances and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (UN, 2011), and it was therefore endorsed as scientifically valid for both purposes. The test substance TM 09 -218 elicited an In Vitro Irritancy Score of 2.8 for ocular irritation and the corneas were noted as clear. It was concluded that TM 09-218 was not classified for ocular irritation.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted on the target substance, in an appropriate test model and according to internationally redognised guidelines.

Justification for selection of eye irritation endpoint:
The study was conducted on the target substance, in an appropriate test model and according to internationally redognised guidelines.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin corrosion / irritation

Skin corrosion is defined as the production of irreversible damage to the skin following application of the test substance. Skin irritation is the production of reversible damage to the skin following application of the test substance. Substances can be allocated to one of two categories based on corrosive effects on the skin (Category 1) and irritating to the skin (Category 2) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

 

Two in vitro tests were performed with the test substance, TM 09-218. The first was an in vitro EpiDerm™skin corrosion test using reconstructed human epidermis. The results of this type of test indicate that where the tissue viability from the 3 minute exposure is less than 50 % of the negative control value, then the test material is classified as corrosive. If the tissue viability from the 3 minute exposure is greater than or equal to 50 % of the negative control value but the 1 hour value is less than 15 % of the negative control value, the test material is classified as corrosive. If the tissue viability from the 3 minute exposure is greater than or equal to 50 % and the 1 hour value is greater than or equal to 15% of the negative control value, then the test material is classified as non-corrosive.

The test substance, TM 09-218, elicited a mean tissue viability of 94.7 % for three minute contact and 80.5 % for one hour contact and was therefore considered non-corrosive.

 

The second in vitro test performed on the test substance, IFF TM 09-218, was the EpiSkin™ Skin Irritation Test using reconstructed human epidermis skin constructs. The results of this type of test indicate that where the mean tissue viability is equal to or less than 50 % of the negative control value, the sample is classed as a Category 2 irritant (GHS classification). The test substance, TM 09-218, elicited a mean tissue viability of 23.9 ± 7.7 % and was therefore classified as a skin irritant (Category 2).

 

Eye irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay or vision following application of the test substance to the anterior surface of the eye which is not fully reversible. Eye irritation means the production of changes in the eye following application of the test substance which is fully reversible. Substances can be allocated to one of two categories based on irreversible effects on the eye (Category 1) and irritating to the eye (Category 2) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

 

An in vitro test was performed using the Bovine Corneal Opacity and Permeability Assay. In this type of test, an in vitro irritancy score (IVIS) ≤3 results in no classification as an eye irritant. An IVIS score of >3; ≤ 55 indicates that no prediction can be made. An IVIS score of >55 results in a classification as a Category 1 eye irritant. The test substance, TM 09-218, elicited an In Vitro Irritancy Score of 2.8 ± 1.2 indicating that the test substance is not an eye irritant.