Butanal, 4-(heptyloxy)-3-methyl-

Endpoint summary

Description of key information

The skin corrosivity of the test substance, TM 09-217, was determined according to OECD Test Guideline 431 using the Reconstructed Human Epidermis Model. The test substance TM 09-217 was classified as non-corrosive to the skin. 
The skin irritation potential of the test substance, TM 09-217, was positive according to OECD Test Guideline 439 using the In Vitro Skin Irritation: Reconstructed Human Epidermis Method. A mean tissue viability of 26.1 ± 3.2%, was predicted and therefore is considered as irritant to the skin. 
The eye irritation potential of the test substance TM 09-217 was assessed in vitro  according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability assay method, indicating that the test substance was not irritating.
The eye irritation potential of the test substance, TM 09-217, was assessed as not an eye irritant according to OECD Test Guideline 405 using an in vivo 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 17 February 2014 and 24 February 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 the In Vitro Skin Irritation: Reconstructed Human Epidermis 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

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 09-217, 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 09-217, 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: 24 February 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 ± 2ºC in a humidified atmosphere of 5% CO2 in air.

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.
The controls and their results were shared with other studies performed in the same assay.

Preparation/application of samples
The test substance, IFF TM 09-217, a clear liquid, positive and negative controls were in liquid form and were applied by dispensing a volume of 10 µL over 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.

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 ± 2ºC 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 ± 2ºC 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 2-8 ºC, protected from light, for 48 - 70 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. The extractant 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:

26.1

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 viability as percentage of mean OD negative control for the test substance was 26.1 ± 3.2.

Possible reduction of MTT by test substance

There was no change in the test substance, IFF TM 09-217/MTT solution or the water control/MTT solution after three hours incubation in the dark at 37±2°C in a humidified atmosphere of 5% CO₂in air. The test substance had not interacted with the MTT.

 

Check for colouring potential of test substance

The test substance, IFF TM 09-217/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 0.832 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 3.2 which was below the maximum value of 18.

 

Positive control

The percentage mean viability of the positive control was 15.7 ± 3.7 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

Episkin results

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	100.2	103.1	96.7	100.0 ± 3.2	Not applicable
Positive Control	19.9	12.9	14.3	15.7 ± 3.7	Irritant
IFF TM 09-217	22.5	27.2	28.6	26.1 ± 3.2	Irritant

 

EpiSkin study data

Sample	Tissue Replicate	Optical Density (OD)	OD-Blank	% Negative Control
Negative control	a	0.991	0.844	100.2
		0.968	0.822
	b	1.022	0.876	103.1
		0.986	0.840
	c	0.971	0.825	96.7
		0.930	0.784
	Replicates a, b, c	Average	0.832	100.0
		SD	0.030	3.2
Positive control	a	0.322	0.176	19.9
		0.301	0.155
	b	0.260	0.114	12.9
		0.247	0.101
	c	0.267	0.120	14.3
		0.264	0.118
	Replicates a, b, c	Average	0.131	15.7
		SD	0.029	3.7
IFF TM 09-217	a	0.331	0.188	22.5
		0.329	0.186
	b	0.374	0.231	27.2
		0.365	0.222
	c	0.387	0.244	28.6
		0.375	0.232
	Replicates a, b, c	Average	0.217	26.1
		SD	0.024	3.2
Blank for positive and negative control		0.140
		0.151
		0.147
		0.145
		0.145
		0.149
	Average	0.146
	SD	0.004
Blank for IFF TM 09-217		0.138
		0.146
		0.144
		0.139
		0.146
		0.143
	Average	0.143
	SD	0.003

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 09-217, with a mean tissue viability of 26.1 ± 3.2%, was predicted as irritant to the skin.

Executive summary:

The skin irritation potential of the test substance, TM 09-217, was positive according to OECD Test Guideline 439 using the In Vitro Skin Irritation: Reconstructed Human Epidermis Method. A mean tissue viability of 26.1 ± 3.2%, was predicted and therefore is 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 vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 May 2014 and 14 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 405 using an in vivo method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals for this study were selected from a stock supply of healthy adult rabbits of the New Zealand White strain. They were in the weight range of 2.86 to 3.05 kg and 34 or 39 weeks of age, prior to treatment (Day 1). All rabbits were acclimatised to the experimental environment for a period of 8 to 15 weeks prior to the start of the study.

Each animal was housed individually in a plastic cage with perforated floors and was offered 125 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum. The batch of diet used for the study is analysed for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. A dietary supplement of hay was offered during acclimatisation until two days prior to dose instillation, for the remainder of acclimatisation and throughout the study observation period wholemeal bread was offered.

During the acclimatisation and study period the animals were given small soft white untreated wood blocks for environmental enrichment.

Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available to Huntingdon Life Sciences. Animal room environmental controls were set to maintain temperature within the range 16 to 20°C, and relative humidity within 40 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period.

Each animal was identified by a numbered tag placed through the edge of one ear. This identification was unique within the Department throughout the duration of the study. Each cage was identified by a coloured label displaying the study number and animal number.

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

Single ocular dose

Observation period (in vivo):

1, 24, 48 and 72 hours and eight days after treatment

Number of animals or in vitro replicates:

Three

Details on study design:

Topical anaesthetics and systemic analgesia:
The following topical anaesthetic and systemic analgesia regime was employed:
60 minutes before administration: 0.01 mg/kg buprenorphine was administered subcutaneously.
15 minutes before administration topical anaesthesia was administered: one/two drops of topical ocular anaesthesia were applied to each eye; the procedure was repeated 5 minutes later. The test material was administered approximately 10 minutes after the last administration of anaesthesia.
Eight hours after test material administration: 0.01 mg/kg buprenorphine and 0.5 mg/kg meloxicam were administered subcutaneously.
Twenty-four hours after instillation of the test substance the second and third animalstreated received 0.5 mg/kg meloxicam administered subcutaneously.

Treatment Procedure:
The eyes of each animal were examined prior to instillation of the test substance to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation. Each animal was gently restrained. The dose was instilled into the right eye by pulling the lower eyelid away from the eye ball to form a cup into which the test substance was dropped. The eyelids were then gently held together for one second before releasing; the left eye
remained untreated.
A single animal (Number 90) was treated in advance; in the absence of a severe effect in this animal two further animals were committed to the study.

Serial Observations
Clinical Signs:
The behaviour of each rabbit was observed immediately following instillation of the test substance to allow assessment of the initial pain response. The animals were returned to their cages and checked at least twice during the first hour after dosing and at regular intervals throughout the day to ensure no severe injury passed unnoticed. Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours and eight days after treatment.

Ocular Responses:
The untreated eye was used as a comparison with the treated eye during assessment of ocular lesions.

Termination:
Following completion of the observation period the animals were humanely killed by an intravenous injection of sodium pentobarbital.

The presence or absence of ulceration or necrosis was designated + (positive) or - (negative).
An opthalmoscope or pencil beam torch was available for use to facilitate inspection of the eyes.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

Animal 90F #

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: # = Sentinel animal; F = Female

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

Animal 91F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: F = female

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

Animal 92 F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: F = Female

Irritation parameter:

iris score

Basis:

mean

Remarks:

Animal 90F #

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Remarks on result:

other: # = Sentinel animal; F = Female

Irritation parameter:

iris score

Basis:

mean

Remarks:

Animal 91F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Remarks on result:

other: F = Female

Irritation parameter:

iris score

Basis:

mean

Remarks:

Animal 92F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Remarks on result:

other: F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Redness)

Basis:

mean

Remarks:

Animal 90F#

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

3

Remarks on result:

other: # = Sentinel animal; F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Redness)

Basis:

mean

Remarks:

Animal 91F

Time point:

other: 24, 48 and 72 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Redness)

Basis:

mean

Remarks:

Animal 92F

Time point:

other: 24, 48 and 72 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Chemosis)

Basis:

mean

Remarks:

Animal 90F #

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: # = Sentinel animal; F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Chemosis)

Basis:

mean

Remarks:

Animal 91F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: F = Female

Irritation parameter:

conjunctivae score

Remarks:

(Chemosis)

Basis:

mean

Remarks:

Animal 92F

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: F = Female

Irritant / corrosive response data:

Ocular Responses
Injection of the conjunctival blood vessels and moderate discharge were evident in the sentinel animal one hour after instillation. Injection of the conjunctival blood vessels, very-slight chemosis and slight or moderate discharge were apparent in the remaining animals at this time; the conjunctival injection persisted throughout the first 24 hours after instillation in these animals. The treated eye of each animal was overtly normal 48 hours after instillation. Instillation of the test material gave rise to practically no initial pain response

Other effects:

Clinical Signs
There was no sign of toxicity or ill health in any rabbit during the observation period.

Mean Values for ocular lesions for Kay and Calandra Classification

Mean irritation scores after instillation of TM 09-217

Area of Eye	1 hour	24 hours	48 hours	72 hours
Cornea	0.0	0.0	0.0	0.0
Iris	0.0	0.0	0.0	0.0
Conjunctiva	6.7	1.3	0.0	0.0
Total Mean Score	6.7	1.3	0.0	0.0

 

Mean Values for ocular lesions for EC (Regulation 1272/2008) and GHS Classification

24, 48 and 72 hours after installation of TM 09-217

Animal No. and Sex	Corneal Opacity	Iridial lesions	Redness of Conjunctiva	Chemosis
90F	0.0	0.0	0.0	0.0
91F	0.0	0.0	0.3	0.0
92F	0.0	0.0	0.3	0.0
F = Female

Grades for ocular irritation responses following installation of TM 09-217

Animal Number and Sex: 90F#			Pain evaluation response: 1
Region of the eye	Response	Grade of response at time after instillation (Hours)
		1	24	48	72	#= Sentinel animal   F = Female
Cornea	Opacity (A)	0	0	0	0
	Area (B)	0	0	0	0
	Ulceration	-	-	-	-
	Stippling	-	-	-	-
Corneal Score (A x B x 5)		0	0	0	0
Iris	Value (C)	0	0	0	0
Iridial Score (C x 5)		0	0	0	0
Conjunctiva	Redness (D)	1	0	0	0
	Chemosis (E)	0	0	0	0
	Discharge (F)	2	0	0	0
	Necrosis	-	-	-	-
	Ulceration	-	-	-	-
Conjunctival Score ((D+E+F) x 2)		6	0	0	0

Grades for ocular irritation responses following installation of TM 09-217

Animal Number and Sex: 91F			Pain evaluation response: 1
Region of the eye	Response	Grade of response at time after instillation (Hours)
		1	24	48	72	F = Female
Cornea	Opacity (A)	0	0	0	0
	Area (B)	0	0	0	0
	Ulceration	-	-	-	-
	Stippling	-	-	-	-
Corneal Score (A x B x 5)		0	0	0	0
Iris	Value (C)	0	0	0	0
Iridial Score (C x 5)		0	0	0	0
Conjunctiva	Redness (D)	1	1	0	0
	Chemosis (E)	1	0	0	0
	Discharge (F)	1	0	0	0
	Necrosis	-	-	-	-
	Ulceration	-	-	-	-
Conjunctival Score((D+E+F)x2)		6	2	0	0

Grades for ocular irritation responses following installation of TM 09-217

Animal Number and Sex: 92F			Pain evaluation response: 1
Region of the eye	Response	Grade of response at time after instillation (Hours)
		1	24	48	72	F = Female
Cornea	Opacity (A)	0	0	0	0
	Area (B)	0	0	0	0
	Ulceration	-	-	-	-
	Stippling	-	-	-	-
Corneal Score (A x B x 5)		0	0	0	0
Iris	Value (C)	0	0	0	0
Iridial Score (C x 5)		0	0	0	0
Conjunctiva	Redness (D)	1	1	0	0
	Chemosis (E)	1	0	0	0
	Discharge (F)	2	0	0	0
	Necrosis	-	-	-	-
	Ulceration	-	-	-	-
Conjunctival Score ((D+E+F)x2)		8	2	0	0

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The highest total mean score was 6.7 occurring at the one hour observation; accordingly under the criteria Kay and Calandra (1962) TM 09-0217 was classified as “minimally irritating” to the eye. TM 09-0217 did not require labelling in accordance with European Commission regulation 1272/2008.

Executive summary:

The eye irritation potential of the test substance, TM 09-217, was assessed as not an eye irritant according to OECD Test Guideline 405 using an in vivo 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 EPISKIN^TMin vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives (Bothamet al.,1995, Barrettet al., 1998 and Fentemet al., 1998). At its 10^thMeeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKIN^TMmodel as scientifically validated for use as a replacement for the animal test. The EPISKIN^TMmodel is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

 

The EPISKIN^TMmodel is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

 

The procedure followed is based on the recommended EpiSkin^TMSkin Corrosivity Test protocol INVITTOX No118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN^TMmodel. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

An in vitro skin corrosion test was performed on the test substance, TM 09-217. The in vitro EpiDerm^TMskin corrosion test using reconstructed human epidermis 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 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 cm²inserts to form a multilayered, 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 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 substance, IFF TM 09-217, elicited a mean tissue viability of 120.7% for three minute contact and 104.3 % 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 -217. The EpiSkin^™Skin Irritation Test using reconstructed human epidermis skin constructs 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 multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm².

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 substance, IFF TM 09-217, elicited a mean tissue viability of 26.1 ± 3.2%, for 15 minutes exposure 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 -217 elicited an in vitro irritancy score of 0.7 for ocular irritation and the corneas were noted as clear. It was concluded that TM 09-217 was not classified for ocular irritation..

An in vivo study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit and was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012).

Injection of the conjunctival blood vessels and moderate discharge were evident in the sentinel animal one hour after instillation. Injection of the conjunctival blood vessels, very-slight chemosis and slight or moderate discharge were apparent in the remaining animals at this time; the conjunctival injection persisted throughout the first 24 hours after instillation in these animals. The treated eye of each animal was overtly normal 48 hours after instillation. Instillation of the test material gave rise to practically no initial pain response. A mean conjuctival score (redness) of 0.33 was observed in two animals. In all other irritation parameters the score was 0.0 in all animals and was therefore TM 09-217 was not classified as an ocular irritant.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted in vitro in an appropriate test species according to internationally recognised guidelines.

Justification for selection of eye irritation endpoint:
The study was conducted in vivo in an appropriate test species according to internationally recognised 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 09-217. The first was an in vitro EpiDerm^TMskin 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 09-217, elicited a mean tissue viability of 120.7% for three minute contact and 104.3 % for one hour contact and was therefore considered non-corrosive.

The second in vitro test performed on the test substance, IFF TM 09-217, was theEpiSkin^™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 09-217, elicited a mean tissue viability of 26.1 % 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 -217, elicited an In Vitro Irritancy Score of 0.7 ± 0.5 indicating that the test substance is not an eye irritant.

An in vivo test was also performed using the acute eye irritation/corrosion assay. In this type of test a category 1 irreversible effect occurs, if, when applied to the eye of an animal, a substance produces at least in one animal effects on the cornea, iris or conjuctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days and/or in at least in 2 of 3 tested animals, a positive respone of corneal opacity ≥ 3 and/or iritis >1.5 calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

For a category 2 reversible effect, if, when applied to the eye of an animal, a substance produces at least in 2 of 3 tested animals a positive response of corneal opacity ≥1 and/or iritis ≥1, and /or conjunctival redness ≥2 and/or conjunctival oedema (chemosis) ≥2 calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, amd which fully reverses within an observation period of 21 days.

The test substance, TM 09-217, elicited a mean conjuctival redness score of 0.33 in two animals and is therefore not classified as an irritant.