Isoleucine, N-(1-oxohexadecyl)-

Endpoint summary

Description of key information

The skin irritation potential is assessed according to a vitro test OECD 439.
The eye irritation potential is evaluated based on a weight of evidence strategy (OECD test n°437 and a vivo test OECD405 from a read-across substance)

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:

2009-09-22 to 2009-09-30

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD guideline for the testing of chemicals, draft proposal for a new guideline, "In Vitro Irritation: Human Skin Model": reconstructed Human Epidermis (RHE) Test Method (20 March, 2099, version 6, 2nd circulation)

Deviations:

not specified

Principles of method if other than guideline:

The prediction model of Skinethic irritation assay is described below.
The test item is considered to be irritant to skin (R38), if the mean relative viability after 42 minutes exposure and 42 hours recovery is less or equal to 50% of the negative control (PBS).

1-Acceptance criteria:
Negative control : Data meet the acceptance criteria if the mean OD570 value of the 3 tissues is >= 1.2. The standard deviation value is considered as valid if it is <= 18%, according to the performance standard (ECVAM SIVS, 2007)
Positive control: Data meet the acceptance criteria if the mean viability, expressed as % of the negative control is <40% and the standard deviation value is <=18%
Batch acceptance criteria: all test item data from one batch are considered as valid if both negative and positive controls data fulfill the above criteria requirements.

II-MTT interference
Preliminary contact experiment was perform with all test item and MTT solution. The test item did not induce a change in colour of the MTT solution to blue or purple appearance. The test item did not interfere with MTT.

III-MTT viability testing
Data were valid, in accordance to the above criteria, Test item did not induced a decrease of viability below 50%.
Negative control OD : 1,669-1,631-1,694
Positive control OD : 0,027-0,019-0,023
Test Item OD : 1,898-1,439-1,798
Viability : 102,81%
SD : 14,51
IL-1alpha mean release : 57,9 pg/mL
Folowwing the irritation prediction model, test item is not irritant

GLP compliance:

yes

Species:

other: not applicable (in vitro)

Strain:

other: not applicable (in vitro)

Type of coverage:

other: not applicable (in vitro)

Preparation of test site:

other: not applicable (in vitro)

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro) : Epidermis negative control: concurrent no traitment Epidermis positive control : SDS 5% in water

Amount / concentration applied:

16 mg (+/- 2 mg)/Epidermis

Duration of treatment / exposure:

42minutes (+/- 1 minute)

Observation period:

42hours (+/-1h) after the end of exposure

Number of animals:

not applicable (in vitro)
3 Epidermis/condition

Details on study design:

I-MTT interference of the test item:
In order to assess if the test item interferes with MTT, a preliminary contact experiment was performed. A 24-well plate was filled with 300 µl of MTT (1mg/mL). 16 mg of the test item was added and incubated at 37°C for 3hours. Whether the MTT solution colour has become blue or purple, which means that the product interact with MTT, it would has been necessary to evaluate, the part of OD due to the non specific reduction of MTT, by using killed epidermis.

II- Tissue treatment:
The day before testing, the Skinethic RHE reconstructed epidermis were placed onto 1 mL of maintenance medium in 6-well plates. 10µL of distilled water were spreaded to the epidermis surface, in order to improve further contact between the solid test item and the epidermis. The 16mg (+/-2mg) of the test item were applied to the epidermis surface without addition of a nylon mesh, on 3 in vitro reconstructed human epidermal tissues. Tissues were then incubated for 42 minutes (+/- 1 minute) at room temperature, in 300µL of maintenance medium. For each test item, a single 24-well plate was used, to avoid cross-contamination between tissues. At the end of the incubation period, the tissues were rinsed with 25 mL of PBS, transferred in a new 24-well plate and allowed to recover for 42h at 37°C. Negative and positive control compounds were applied in parallel (PBS and SDS 5%). After 42 h (+/- 1 hour) of recovery, the cell viability was evaluated using the MTT test (n=3). Culture medium was collected for ELISA.

III-MTT assay procedure
In a 24-well plate, a volume of 300 µL of MTT solution at 1 mg/mL in assay culture medium was added to all test wells containing tissue or blank and incubated for 3 h (+/- 5 minutes) at 37°C (5%CO2). After incubation, the MTT solution was discarded and tissues were placed on absorbent parper. The epidermis were then placed in a new 24-well plate containing 800 µL of isopropanol. 700 µL of isopropanol were then added on the top of the tissue, which were then incubated at room temperature for 2h (+/- 5 minutes). A volume of 3X200 µL aliquots of blue MTT extraction solution was transferred from each well to corresponding wells of a 96-well plate. The Optical Density at 570 nm was determined spectrophotometrically using a microplate reader. The mean OD570 of the untreated control tissues exposed to PBS was set to represent 100% of viability and the results were expressed as a percentage of these control.

IV-IL-1alpha Quantification:
Tissue supernatants were harvested 42 hours after test item application. They were stored at -20°C untill analysis. Culture medium samples were processed for IL-1alpha detection by using ELISA, according to the kit manufacturer's procedure (Pierce).

Irritation / corrosion parameter:

other: other: Viability-MTT

Value:

> 100

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 42 hours. Reversibility: other: non applicable. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the irritation prediction model, the test item is not irritant

Executive summary:

Dermal acute irritation is a local, reversible inflammatory response of normal living skin to direct injury caused by the application of an irritant substance.The assesment of the irritant potential of test items in vitro is done using validated in vitro skin irritation assay such aas Skinethic RHE assay. It discriminates irritants (R38) and non-irritant chemicals, on the basis of reduction in viability under 50% after application of test item. The protocol used in this study is based on the OECD guideline for the testing of chemicals, draft proposal for a new guideline, "in vitro skin irritation: Human Skin Model Test".

After 42 minutes of application of test item and reference on the top of the epidermis and recovery for 42 hours, tissue viability was determined by MTT viability testing. In these conditions, test item did not decrease the viability of the epidermis, suggesting that according to the irritation prediction model, the test item is not irritant

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-09 to 2010-04-12

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD 437

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

other: Mineral oil

Amount / concentration applied:

750 µL/cornea test item at 20% in vehicle

Duration of treatment / exposure:

4 hours ± 10 minutes

Observation period (in vivo):

After rinsing

Number of animals or in vitro replicates:

no animal

Details on study design:

Biological system preparation

Eyes selection:
detection of defects of the cornea (neovascularization, pigmentation, opacity, scratches) by immersing the eyes in a jar containing buffered Hanks medium and putting them under lighting.

Dissection and immersion of corneas:
for each selected eye
- incision on the scleral ring, using a scalpel, then dissection of the cornea, using scissors, leaving 2 or 3 mm of the scleral ring
- immersion of the corneas in buffered Hanks medium, the epithelial side upward. - use of the "fresh" corneas.

Mounting of the holders:
- deposit of each fresh corneas, epithelial side upward, on the posterior part of the holders
- placement of the anterior part of the holder above the corneas
- screwing on with an electric screwdriver in order to ensure the waterproofness of the system without any risk for the cornea
- identification of each cornea with the corresponding holder number.

Pre-incubation of the corneas:
- in order to eliminate the air bubbles, filling, excessively, of the anterior compartments (epithelial side) and posterior (endothelial side), with nutritive medium at room temperature
- incubation of the holders in a water-bath at 32 ± 1 °C, for at least one hour, in a horizontal position and immersed in three quarters of their
height.

Measurement of the initial opacity of the corneas

- realised using an OP KIT opacitometer, which determines a change in light transmission between an "empty" holder (containing nutritive medium only) and a "treated" holder, and displays a numerical value of opacity.
- calibration of the opacitometer, with specific calibrators, before the measurement of the corneal opacity
- values of opacity of the calibrators:
- calibrator 1: 73 to 77
- calibrator 2: 147 to 153
- calibrator 3: 220 to 240
- if necessary: adjustment with the "calibrator" button
- change of the nutritive medium of the posterior compartment
- wiping of each holder
- measurement of the initial opacity (OPT0) of each cornea versus an "empty" holder (containing only nutritive medium).

Preparation and modalities of application of controls ant test item

Carrying out in the same chronological order of each operation (treatment, rinsing and readings), by series of 3 corneas.
- negative control: Sodium Chloride to 0,9% (W/V) (NaCl – CAS N° 7647-14-5)
- vehicle control: mineral oil (CAS N° 8042-47-5)
- positive control: 20% imidazole solution (CAS N° 288-32-4) (W/W) in NaCl to 0.9%
- measurement of the pH of each control
- control of the homogeneity and stability of each control in the trial conditions
- test item:
- measurement of the pH of the test item and the preparation
- control by I.E.C. of the homogeneity of the test item and confirmation by the Sponsor of the stability in the storage conditions
- extemporaneous preparation of the test item: to be diluted to 20% (W/W) in mineral oil
- control of the homogeneity and stability of the preparation in the trial conditions

- aspiration of the nutritive medium of the anterior compartment using a specific vacuum pump
- remove the nut and the glass disk
- application of 750 µL of the preparation and of each control on the cornea using a micropipette
- starting of the chronometer for each series, after the treatment of the 1st cornea
- incubation in a water-bath at 32 ± 1 °C, in a vertical position (screw upward) for 4 hours ± 10 minutes.

Rinsing (at the end of the contact timepoint)

- aspiration of each control and of the preparation
- rinsing at least 2 times with nutritive medium with phenol red at 32 ± 1°C: the corneas are rinsed more than 2 times if the phenol red is still discolored or the test substance is still visible
- elimination of the excess product which had stuck to the walls of the support with a cotton bud
- final rinsing with nutritive medium
- filling of the anterior with nutritive medium at 32 ± 1 °C.

Measurement of the corneal opacification

- replacing of the nutritive medium in the posterior compartments using a vacuum pump
- wiping of each holder
- measurement of the opacity OPT2 of each cornea versus an "empty" holder.


Measurement of the corneal permeability

Addition of the fluorescein solution:
- aspiration of the anterior compartment using a vacuum pump
- deposit of 1 mL of the fluorescein solution (0.5%), in the anterior compartment
- incubation of the holders at 32 ± 1 °C for 90 ± 5 minutes in a vertical position.

Measurement of optical density:
- sample of the medium contained in each posterior compartment
- deposit of each medium in tubes identified by the holder number
- transfer of 360 µL of this medium onto microplates of 96 wells
- measurement of the optical density (O.D.) with a spectrophotometer at 490 nm (blank = nutritive medium).

Determination of the theoretical O.D. :
Determined for each new batch of fluorescein by establishing a standard curve connecting the O.D. to the concentration of fluorescein (1-2-3-4-5-6-7-8-9-10-12-14-16-20-40 µg/mL). The graph being under a linear form, the calculation of the gradient permitted the calculation of theoretical O.D. for the concentration at 10 µg/mL.

It also permitted to determine a value of optical density beyond which the medium had to be diluted (beginning of the plate stage). Each medium of which the O.D. exceeded this value was diluted to 1/4 in the nutritive medium. The O.D. value obtained was then multiplied by 4, during the final calculations.


Corneas examination

- dismantling of each holder
- observation of the cornea condition: epithelium detachment, visible modification of the cornea (oedema, colouring, …).

Calculation

- calculation of the means of each parameter (OPT2-OPT0 and O.D.)
- correction of the values by subtracting the negative controls corneas
- calculation of the in vitro irritancy score (IVIS) from the corrected values:

IVIS = (OPT2-OPT0) + (15 x O.D.)

Irritation parameter:

other: measurement of corneal opacification and permeability, and optical density

Run / experiment:

mean / of each parameter measured

Value:

24.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: See the method in the section "details on study design"

Irritant / corrosive response data:

Score (4 hours): 24,0 ± 1,7
Classification: Test item not classified corrosive or severe irritant

Interpretation of results:

other: Test item not classified corrosive or severe ocular irritant

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Test item not classified corrosive or severe ocular irritant

Executive summary:

The objective of this test was to evaluate the ocular primary irritation of a chemical substance by the determination of its effect on the opacity and permeability of bovine corneas obtained from eyes freshly collected in a slaughter-house. This test is an In vitro study performed on the isolated bovine cornea, according to the OECD protocol n° 437.

The bovine corneal opacity and permeability method (BCOP) is based on the Muir method (1984, 1985). This technique is adapted from the one described by Gautheron and coll., 1992 to determine the ocular irritant potential of diverse substances.

Thanks to this method, two parameters such as corneal opacity and permeability can be measured.

Under the experimental conditions adopted, the test item diluted at 20% (W/W) in mineral oil, basing on the score obtained, will not be classified as corrosive or severe ocular irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Concerning the eye irritation potential, a vivo test OECD 405 performed on a read-across substance demonstrated that this structurally closed substance was highly irritating to the eyes (R41), but a vitro test OECD 437 performed on the test item gave a négative response meaning that the test item is not highly irritant to eyes. So the test item was classified Eye irritant category 2 taking into account the available data mentionned above.

Justification for selection of skin irritation / corrosion endpoint:
The assesment of the irritant potential of test items in vitro is done using validated in vitro skin irritation assay such as Skinethic RHE assay. It discriminates irritants (R38) and non-irritant chemicals, on the basis of reduction in viability under 50% after application of test item. The protocol used in this study is based on the OECD guideline for the testing of chemicals, draft proposal for a new guideline, "in vitro skin irritation: Human Skin Model Test".

Justification for selection of eye irritation endpoint:
The classification related to eye irritation is based on a weight of evidence strategy taking into account data from an in vitro test OECD 437 performed on the test substance and data from a vivo test OECD 405 performed on a read-across substance. No validated in vitro test is available to assess the eye irritation potential of a substance.

Effects on eye irritation: irritating

Justification for classification or non-classification

The test item is classified Eye irritant category 2 H319 but not classified for skin irritation according to the CLP regulation 1272/2008/EC.