Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
28 August 2101 to 03 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, guideline study, available as an unpublished report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Physical state: Pale Yellow Solid
- Purity: Not applicable - UVCB
- Substance identity: Aluminum, benzoate C16-18-fatty acids complexes
- Batch number: 11074091 + Benzoic acid
- Carbon Content: 65.1%
- Analysis code: A118
- Date recieved: 07 June 2012
- Expiration date: 01 July 2013
- Storage of test material: Room temperature in the dark

Test animals

Species:
other: EPISKIN (TM) reconstructed human epidermis model
Strain:
other: Not applicable
Details on test animals and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Not applicable
Amount / concentration applied:
- Treatment group: Test carried out in triplicate. Approximately 10 mg of the test item was applied topically, ensuring an even covering, to the epidermis surface which had previously been moistened with 5 µL sterile, distilled water to improve contact between the solid test item and the epidermis.
- Negative control: 10 µL Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++
- Positive control: 10 µL Sodium Dodecyl Sulphate (SDS) at 5% w/v aqueous solution spread over entire surface of the epidermis using a pipette tip with the process being repeated after 7 minutes.
Duration of treatment / exposure:
- Treatment period: 15 minutes
- 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. 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.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
Triplicate samples of epidermis tissue, previously moistened with 5µL sterile distilled water to improve contact with the solid test item, were uniformly covered with approximately 10 mg of test item. At the end of the 15 minute exposure period, each tissue was removed and rinsed with DPBS with Ca++ and Mg++ . The tissues were then incubated for 42 hours at 37°C and 5% CO2 in air, in wells containing 2 mL maintenance media.

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 37ºC, 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 10ºC 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:
Value:
98.4
Remarks on result:
other:
Remarks:
Basis: mean. Time point: After a 15-minute exposure. Max. score: 101.1. Reversibility: no data. Remarks: Negative control item (set at 100). Standard deviation relative to mean viability ± 3.4.. (migrated information)

In vivo

Irritant / corrosive response data:
- Viability: The relative mean (n = 3) viability of the treated tissues was 98.4 after a 15-minute exposure period, with standard deviation of 3,4%
- Optical density: The mean optical density of the treated tissues at 540 nm was 0.777
- Conclusion: Test item is considered to be non-irritant using the EPISKIN (TM) human epidermis model (viability >50%)
Other effects:
- 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.

Any other information on results incl. tables

-Direct MTT reduction: The MTT solution containing the test item did not turn blue, indicating that test item did not directly reduce MTT.

The acceptance criteria were 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 3.4 % and hence the test item acceptance criterion (≤ 18%) was also satisfied.

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 Item1

0.843

0.790

0.046

106.7

100*

5.5

0.758

97.2

0.760

96.2

Positive Control Item1

0.119

0.093

0.030

15.1

11.8

3.8

0.100

12.7

0.060

7.6

Test Item

0.785

0.777

0.027

99.4

98.4

3.4

0.747

94.8

0.799

101.1

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:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Aluminum, benzoate C16-18-fatty acids complexes is considered to be non-irritant using the EPISKIN (TM) human epidermis model.
Executive summary:

Aluminum, benzoate C16-18-fatty acids complexesis 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.