Allyl heptanoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-14 to 2009-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B.46

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the testing of chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 9 September 2009, 3rd WNT circulation, Vers. 7.6.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Allyl heptanoate

EC Number:

205-527-1

EC Name:

Allyl heptanoate

Cas Number:

142-19-8

Molecular formula:

C10H18O2

IUPAC Name:

allyl heptanoate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

15 µL of the undiluted test item were applied to each of triplicate tissues.
For the positive and negative controls 15 µL were dosed per tissue.

Duration of treatment / exposure:

15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5% CO2

Duration of post-treatment incubation (if applicable):

After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.

Number of replicates:

three replicate tissues with two replicate wells each per exposure and control group

Test system

Details on study design:

CELL CULTURE:
EpiSkin TM kits (Lot No.: 09-EKIN-035) are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.

TREATMENT:
The negative control (deionised water (lot no. 091009; 15 µL was applied to each of triplicate tissues) and positive control (5% sodium lauryl sulphate (Fluka, batch no. 1353471 51508322) solution in deionised water; 15 µL was applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin TM triplicate tissues. The plates were placed into the incubator for 15+/- 1 min at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.
After the end of the treatment interval the inserts were removed immediately from the plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 +/- 1 hour at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4, 5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues.The percent reduction of cell viablity in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure (42 hours) was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 +/- 1.5 °C, 5 +/- 0.5 % CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Tissues samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 70 hours in the refrigerator at 2-8 °C.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax© Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38, category 2 is predicted if the mean relative tissue viability of three individual tissues is reduced below or equal to 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥0.6 till ≤ 1.5.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
In case the standard deviations in between tissues of the same treatment group is ≤ 18%.

TEST FOR DIRECT MTT REDUCTION.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 µL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
A colour change could not be observed.
No further information on the study design was stated.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three tests with two replicate wells each

Value:

>= 54.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

5% SLS (Sodium lauryl sulphate)

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Results after treatment with the test substance

 

Dose group	Treat-ment Interval	Absor-bance 570 nm Tissue 1	Absor-bance 570 nm Tissue 2	Absor-bance 570 nm Tissue 3	Mean Absor-bance of 3 Tissues	Absorbance [%] Tissue 1, 2 + 3	Standard Deviation [%]	Rel. Absorbance [%] of Negative Control]
Negative Control	15 min	1.2513	1.0690	1.2215	1.1806	106 91 104	8.3	100.0
Positive Control	15 min	0.3342	0.4393	0.5269	0.4335	28 37 45	8.2	36.7*
Test Item	15 min	0.6414	0.5758	0.7288	0.6486	54 49 62	6.5	54.9

* The validity of the test system was not influenced by the fact, that the positive control absorbance value is above the range of the historical data, since the "OECD Guideline for the Testing of Chemicals, Draft Proposal for a New Guideline, In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, 9 September 2009, 3rd WNT circulation, Vers. 7.6" only demands a clear positive effect (≤40%) on the tissues after exposure to the positive control

 

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

 

Historical data:

Positive Control		Negative Control
Number of Studies	48	Number of Studies	48
Period	March 2009 – March 2010	Period	March 2009 – March 2010
Mean Viability	17.0%	Mean Viability	1.063
Standard Deviation	11.0%	Standard Deviation	0.176
Range of Viabilities	6% - 28%	Range of ODs	0.8 – 1.3

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

After treatment with the test item Allyl heptanoate (Sym09/611041) the relative absorbance values were decreased to 54.9 %. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
The test item should not be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.