Oxoaluminium, C16-C18-alkyl esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2101 to 03 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(octadecanoato-O)oxoaluminium

EC Number:

236-521-7

EC Name:

(octadecanoato-O)oxoaluminium

Cas Number:

13419-15-3

Molecular formula:

C18H35AlO3

IUPAC Name:

(octadecanoato-kappaO)(oxo)aluminum

Test material form:

liquid: viscous

Details on test material:

- Physical state: Pale yellow viscous liquid
- Substance tested: 50% w.w. concentration in pharmaceutical white oil (CAS No. 8042-47-5).
- Batch number: 11074091
- Carbon Content: 74.73 %
- Analysis code: Report No. 22349
- Date recieved: 15 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not reported

Source strain:

not specified

Details on animal used as source of test system:

- Source: EPISKIN (TM) model kit
- Supplier: Skin Ethic Laboratories, Nice, France

Justification for test system used:

The EPISKIN model is a three-dimernsional 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 of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Vehicle:

unchanged (no vehicle)

Details on test system:

The study was carried on a 50% w.w. concentration of the test item in pharmaceutical white oil.
To make sure that the test item did not directly reduce the MTT to blue formazan, 10 µL of the test item 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°C and 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. As the MTT solution containing the test item did not turned blue the test item was shown not to directly reduce MTT.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Treatment group: Test carried out in triplicate. 10 µL of the test item was applied topically, ensuring an even covering, to the epidermis surface.

Duration of treatment / exposure:

- Treatment period: 15 minutes

Duration of post-treatment incubation (if applicable):

- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a soft stream of DPBS to gently remove any residual test item. Residual test item remained on the tissue surface after rinsing. The rinsed tissues were transferred to a second column of three wells, each containing 2 mL of maintenance medium, and incubated for 42 hours, at 37°C and 5% CO2 in air.

Number of replicates:

Triplicate samples of epidermis tissue were uniformly covered with approximately 10 µL of test item. At the end of the 15 minute exposure period, each tissue was removed and rinsed with DPBS with Ca++ and Mg++. Residual test item remained on the tissue surface after rinsing. The tissues were then incubated for 42 hours at 37°C and 5% CO2 in air, in wells containing 2 mL maintenance media.

Test system

Details on study design:

The measurement of tissue viability (cytotoxicity) was measured by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (by the mitochondrial succinate dehdroganase in viable cells) in the test item treated tissue relative to the negative control using the following procedure. After incubation, the triplicate tissue samples were transferred into three wells each containing 2ml of a 0.3 mg/L MTT solution, care being taken to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air and then the epidermis was carefully separated from the collagen matrix using forceps and both parts placed into 1.5 mL microtubes containing 500 µL of acidified isopropanol. Each tube was plugged and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C for 3 days to allow the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was thoroughly mixed on a vortex mixer to produce a homogeneous coloured solution. The optical density of the extracted solution was measured at 540 nm against an acidified isopropanol blank.
The relative mean viability were calculated using the following equation: Relative mean viability (%) = (mean OD540 of test item/mean OD540 of negative control) x 100. If the mean tissue viability is ≤50%, the substance is considered to be irritant.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Percent Relative Viability

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean. Time point: After a 15 minute exposure. Max. score: 94.9. Reversibility: No data.

Other effects / acceptance of results:

- Remarks: Negative control item (set at 100). Standard deviation relative to mean viability ± 3.0. (migrated information)
- Viability: The relative mean viability of the treated tissues was 93.0 % after a 15-minute exposure period, with standard deviation of 3.0 %.
- Optical density: The mean optical density of the treated tissues at 540 nm was 0.735
- Direct MTT reduction: The MTT solution containing the test item did not turn blue, indicating that test item did not directly reduce MTT.
- Positive control: The relative mean tissue viability was 11.8 % relative to the negative control and the standard deviation was 3.8%
- Negative control: The mean optical density was 0.790 and the standard deviation was 0.046.
- Acceptance criteria: Satisfied according to the protocol criteria for both the positive control (relative mean viability ≤ 40% and standard deviation ≤ 18%) and negative control (OD540 ≥ 0.6 and standard deviation of individual tissue viability ≤18%). The standard deviation of the triplicate treated tissues was 3.0 % and hence the test item acceptance criterion (≤ 18%) was also satisfied.
- Conclusion: Test item is considered to be non-irritant using the EPISKIN (TM) human epidermis model (viability >50%)

Any other information on results incl. tables

Table 1. Mean OD₅₄₀Values and Percentage Viability for Negative Control Item, Positive Control Item and Test Item

Item	OD540of tissue	Mean OD540of triplicate tissues	±SD of OD540	Relative Individual tissue viability (%)	Relative mean viability (%)	±SD of Relative mean viability (%)
Negative Control Item^1	0.843	0.790	0.046	106.7	100*	5.5
	0.758			97.2
	0.760			96.2
Positive Control Item^1	0.119	0.093	0.030	15.1	11.8	3.8
	0.100			12.7
	0.060			7.6
Test Item	0.708	0.735	0.023	89.6	93.0	3.0
	0.747			94.6
	0.750			94.9

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

¹Control group shared with test laboratory project number 41202604

Applicant's summary and conclusion

Interpretation of results:

other: Criteria for classification as a skin irritant not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The substance is considered to be non-irritant using the EPISKIN (TM) human epidermis model.

Executive summary:

The substance is considered to be non-irritant using the EPISKIN (TM) human epidermis model. The skin irritation potential of the test item was evaluated using EPISKIN (TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure period of 42 hours using a colourimetric MTT reduction assay following OECD guideline 439 in an experimental proprietary study (Harlan 2013). The quality criteria required for the acceptance of results in the test were satisfied and the study is considered reliable and relevant for use. The study was carried on a 50% w.w. concentration of the test item in pharmaceutical white oil.