Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-25 - 2018-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

EC Number:

236-109-7

EC Name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Cas Number:

13170-05-3

Molecular formula:

C22H27AlO5

IUPAC Name:

aluminum hydroxide bis(4-tert-butylbenzoate)

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

other: n/a, human

Details on animal used as source of test system:

n/a

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDermTM (MatTek). The EpiDermTM tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25899 for main experiment, 25883 (killed tissues) and 28615 (viable tissues) for additional controls)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 041918TMB, 051718TMP)
1x bottle of DPBS Rinse Solution (Lot No.: 010418ISA, 051518ISA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)
- Tissue batch number(s): Lot No.: 25899 for main experiment, 25883 (killed tissues) and 28615 (viable tissues) for additional controls
- Production date: Lot No.: 25899: 2018-04-25 , 25883: 2018-02-28 and 28615: 2018-05-23
- Date of initiation of testing: 2018-04-25

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: After dosing of all tissues, all tissue plates were transferred to a humidified incubator at 37 ± 1 °C, 5.0% CO2, for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): The tissue plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h at 37 ± 1 °C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: non stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
MTT stock solution: 5 mg/mL MTT (Sigma, Lot MKBZ5197V, VWR; Lot 0977C002) in PBS (Gibco; Lot No.: 1909266, 1943443, 1943446)
MTT medium: MTT stock solution was diluted 1 + 4 with DMEM-based medium (final concentration 1 mg/mL)
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium.
- Incubation time: 60 min at 37 +/- 1 °C
- Spectrophotometer: plate spectrophometer
- Wavelength: 570 nm
- Filter: filter band pass
- Filter bandwidth: maximum +/- 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- N. of replicates : 2
- Method of calculation used: MTT (NSMTT) was calculated relative to negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK]*100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS.
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less or equal to 50% in accordance with regulation EC 1272/2008 (UN GHS “Category 2”). Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50% in accordance with UN GHS “No Category”.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution

Duration of treatment / exposure:

60 ± 5 min

Duration of post-treatment incubation (if applicable):

18 ± 3 h

Number of replicates:

The test was performed on a total of 3 tissues per dose group.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean relative tissue viability

Value:

100.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none stated
- Direct-MTT reduction: Yes
- Colour interference with MTT: The mixture turned blue/purple

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean absolute OD570 of the three negative control tissues was >/= 0.8 and ≤ 2.8 (1.849).
- Acceptance criteria met for positive control: Yes. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.2%).
- Acceptance criteria met for variability between replicate measurements: Yes. Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 2.8%).

Applicant's summary and conclusion

Interpretation of results:

other: not classified based on EU-GHS criteria

Conclusions:

The study was conducted under GLP according to OECD guideline 439 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritating potential of the test substance to the skin in vitro.
The mean relative tissue viability obtained after 60 minutes treatment with the test item and 42 h post-incubation compared to the negative control tissues was 100.9% (NSMTT-corrected). Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. It is concluded that this test is valid and that Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Executive summary:

In the present study the skin irritant potential of Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:

NSMTT [%] = [(OD_KT- OD_KU)/OD_NK] * 100 = -0.1%

Mean OD_KT= 0.051

Mean OD_KU= 0.053

Mean OD_NK= 1.777

NSMTT was ≤30% (-0.1%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TOD_TT) of the test item treated living tissues TM was therefore corrected according to the following formula:

TOD_TT= OD_TM– (OD_KT– OD_KU) = 100.8% - (-0.1%) = 100.9%

The mixture of 25 mg of the test item per 300 µl aqua dest. and per 300 µL isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in water and isopropanol was measured in the range of 570 ± 30 nm.

The test item in isopropanol absorbed light in the relevant range. For quantitative correction of results, the non-specific colour of additional viable tissues (NSC_living) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:

NSC_living[%] = [OD_TVT/OD_NK]*100 = 0.5%

Mean OD_TVT= 0.008

NSC_livingwas ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

As correction with the NSC_livingcontrol was not necessary, correction with the NSC_killedcontrol was also not required.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (100.9%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.

In conclusion the test item showed no irritant effects under the given conditions. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.