Registration Dossier

Administrative data

Description of key information

• Non-irritating to skin (OECD 439)

• Non-irritating to eyes (valid in vitro methods and OECD 405)

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:
2011-11-15 to 2011-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In vitro skin irritation: reconstructed human epidermis test method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The Episkin model 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 loaded with type IV collagen.
Vehicle:
unchanged (no vehicle)
Details on test system:
MTT DYE METABOLISM, CELL VIABILITY ASSAY
- The MTT assay is a colourmetric method of determining cell viability based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
- One limitation of the assay is possible interference of the test material with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if, at the time of the MTT test (after rinsing), there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

TEST FOR DIRECT MTT REDUCTION
- To identify possible interference, the test material was checked for ability to directly reduce MTT.
- 10 μL of test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium.
- The solution was incubated in the dark at 37 ºC, 5 % carbon dioxide in air, for 3 hours.
- Untreated MTT solution was used as a control.
- If the MTT solution containing the test item turns blue, the test material is presumed to have reduced MTT.

EPISKIN MODEL KIT
- Supplier: SkinEthic Laboratories, Nice,.
- Date received: 2011-11-15.

PREPARATION OF NEGATIVE AND POSITIVE CONTROL ITEMS, MTT AND ACIDIFIED ISOPROPANOL
- The negative control item was used as supplied.
- The positive control item was prepared as a 5 % w/v aqueous dilution.
- A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
- A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.
 
PRE-INCUBATION (DAY ZERO: TISSUE ARRIVAL)
- 2 mL of maintenance medium, warmed to approximately 37 ºC, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate.
- Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate).
- A different 12-well plate was used for the test material and control item.
- The tissues were incubated at 37 ºC, 5 % carbon dioxide in air, overnight.
 
MAIN TEST (APPLICATION OF TEST ITEM AND RINSING (DAY 1)
- 2 mL of maintenance medium, warmed to approximately 37 ºC, was pippetted into the second column of 3 wells of 12-well plate.
- Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
- 10 μL of the test item was applied to the epidermis surface.
- Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5 % w/v served as the positive controls.
- To ensure satisfactory contact with the positive control item, the SDS solution was spread over the entire surface of the epidermis using a pipette tip, taking particular care to cover the centre.
- After 7 minutes contact time, the SDS solution was re-spread with a pipette to maintain the distribution of the SDS for the remainder of the contact period.
- The plate(s) were kept in a biological safety cabinet at room temperature for 15 minutes.
- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++.
- Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test material.
- Rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well.
- The rinsed tissues were then incubated at 37 ºC, 5 % carbon dioxide in air, for 42 hours.
 
MTT LOADING AND FORMAZAN EXTRACTION (DAY 3)
- Following the 42 hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium.
- 1.6 mL of maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at minus 14 to minus 30 ºC for possible inflammatory mediator determination.
- 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plate(s).
- Tissues were transferred to the MTT filled wells and care was taken to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper.
- Tissues were incubated for 3 hours at 37 ºC, 5 % carbon dioxide in air.
- At the end of the three hour incubation period, each tissue was placed onto absorbent paper to dry.
- A total biopsy of the epidermis was made using the Episkin biopsy punch.
- The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed.
- Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer.
- The tubes were refrigerated at 1 to 10 ºC until day six of the investigation, allowing the extraction of formazan crystals from MTT-loaded tissues.
 
ABSORBANCE / OPTICAL DENSITY MEASUREMENTS (DAY 6)
- At the end of the formazan extraction period, each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate.
- 200 μL of acidified isopropanol alone was added to the two wells designated as blanks.
- Optical density was measured (quantitative viability analysis) at 540 nm (without reference filter) using an Anthos 2001 microplate reader.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 μL of the test item was applied to the epidermis surface.
Duration of treatment / exposure:
15 minutes.
Duration of post-treatment incubation (if applicable):
42 hours.
Number of replicates:
Triplicate
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean of triplicate runs
Value:
68.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: the test item was non-irritant
Other effects / acceptance of results:
DIRECT MTT REDUCTION
- The MTT solution containing the test item did not turn blue, which indicated that the test material did not directly reduce MTT.
 
TEST MATERIAL, POSITIVE CONTROL AND NEGATIVE CONTROL
- Individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control and positive control are shown in Table 1 (attached).
- Mean viabilities and standard deviation of the test material and positive control relative to the negative control are also given in Table 1 (attached).

The relative mean viability of the test item treated issues was 68.8% after a 15-Minute exposure period.
 
QUALITY CRITERIA
- Relative mean tissue viability for the positive control treated tissues was 4.5 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.4 %. The positive control acceptance criterion was therefore satisfied.
- Mean OD540 for the negative control treated tissues was 0.927 and the standard deviation value of the percentage viability was 8.7 %. The negative control acceptance criterion was therefore satisfied.
- Standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 13.7 %. The test item acceptance criterion was therefore satisfied.
Interpretation of results:
other: not-irritating
Conclusions:
The test material was determined to be non-irritant.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
23 January 2012 to 02 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.23 or 2.26 kg
- Housing: The animals were individually housed in suspended cages.
- Diet: 2930C Teklad Global Rabbit diet available ad libitum.
- Water: Mains drinking water available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes: at least 15 changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light (06:00 to 18:00)
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL of the test item was placed into the conjunctival sac of the right eye. The upper and lower lids were held close together for about 1 second immediately after treatment to prevent loss of the test item, and then released.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
One animal was treated initially. After consideration of the ocular responses produced in the first animal, two additional animals were treated.
Details on study design:
CONTROLS
The left eye remained untreated and was used for control purposes.

REMOVAL OF TEST SUBSTANCE
- Washing: none

SCORING SYSTEM: Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the 6 point scale shown in Appendix 1 (see section "Attached background material"). Assessment of ocular damage/ irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment according to Draize (see Appendix 2 in section "Attached background material")

TOOL USED TO ASSESS SCORE: light source from a standard ophthalmoscope

BODYWEIGHT
Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
(71614)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no corneal effects
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
(71646)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no corneal effects
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: no corneal effects
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
(71614)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no iridial effects
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
(71646)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no iridial effects
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: no iridial effects
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Remarks:
(71614)
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Remarks:
(71646)
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
(71647)
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hrs
Irritant / corrosive response data:
No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Minimal conjunctival irritation was noted in all treated eyes at the 24 hour observation and in two treated eyes at the 48 hour observation.

One treated eye appeared normal at the 48 hour observation and two treated eyes appeared normal at the 72 hour observation.

The individual results are detailed in Table 2.
Other effects:
Bodyweight loss was noted in one animal and two animals showed expected gain in bodyweight during the study.

Table 2: Individual scores for ocular irritation

Rabbit number and sex

71614 Male

71646 Male

71647 Male

IPR = 2

IPR = 2

IPR = 2

Time after treatment

1

hour

24

hours

48

hours

72

hours

1

hour

24

hours

48

hours

72

hours

1

hour

24

hours

48

hours

72

hours

Cornea

Degree of opacity

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

Area

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae

Redness

 

2

 

1

 

0

 

0

 

2

 

1

 

1

 

0

 

2

 

1

 

1

 

0

Chemosis

2

1

0

0

2

1

0

0

2

1

0

0

Discharge

2

1

0

0

2

1

0

0

2

1

0

0

IPR = initial pain reaction

Interpretation of results:
GHS criteria not met
Conclusions:
The test item is considered to be not irritating to the rabbit eye and is not classified in accordance with CLP Regulation No (EC) 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation:

A reliable in vitro study was performed in accordance with GLP and OECD Guideline 439 to evaluate the skin irritation potential of 9-decenoic acid, methyl ester using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Harlan Laboratories Ltd, 2012). The relative mean viability of the test item treated tissues was 68.8 % after the 15 minute exposure period. The test item, 9-decenoic acid, methyl ester was therefore considered to be non-irritant.

In vitro skin irritation studies performed in accordance with OECD Guideline 439 are considered to be valid replacements for the in vivo skin irritation study (OECD Guideline 404). The result of this study on 9-decenoic acid, methyl ester indicates that the test material is not a skin irritant and is considered to be valid without the need to verify the result by performing additional in vivo studies.

Eye irritation:

A reliable in vitro study was performed in accordance with GLP and to accepted scientific standards (Harlan Laboratories Ltd, 2012). The purpose of the study was to determine the eye irritation potential of 9-decenoic acid, methyl ester using the SkinEthic reconstructed Human Corneal Epithelium model after a treatment period of 10 minutes. Triplicate SkinEthic tissues were treated with 30 µL of the test item for 10 minutes. Cytotoxicity was determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues (quantitative measurement of tissue viability) relative to the negative control. The relative mean viability of the test item treated tissues was 113.5% and hence 9-decenoic acid, methyl ester is considered to be a non-irritant. A confirmatory in vivo test was therefore performed.

 

A reliable in vivo study was performed in accordance with GLP and OECD Guideline 405 to assess the irritancy potential of 9-decenoic acid, methyl ester to the eye of the New Zealand white rabbit (Harlan Laboratories Ltd, 2012). A single application of the test item to the non-irrigated eye of three rabbits produced moderate conjunctival irritation. One treated eye appeared normal at the 48 hour observation and two treated eyes appeared normal at the 72 hour observation. Based on the classification criteria for eye irritation according to Regulation (EC) No. 1272/2008, 9-decenoic acid, methyl ester is considered to be non-irritating to eyes.

Justification for classification or non-classification

In the in vitro skin irritation, the in vitro eye irritation and the in vivo eye irritation studies, 9-decenoic acid, methyl ester was non-irritant and therefore requires no classification according to Regulation (EC) No. 1272/2008.