Reaction products of tetraisopropyl titanate (4+), hydroxyalkaloic acid and substituted alkanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 2nd, 2020 to July 3rd, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

Adopted June 18th, 2019

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 6th, 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

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:

TEST SYSTEM SPECIFICATION
The test system is a commercially available EpiDermTM-Kit, procured by MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery:30. Jun. 2020
Batch no.: 30877
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 representing main EpiDermTM lipid classes analogous to those found in vivo. The tissues are cultured on specially prepared cell culture inserts.

CHEMICALS AND MEDIA
- MTT: A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL in the freezer (– 20 ± 5 °C). 2 mL of the stock solution
were thawed and diluted with 8 mL of medium. This MTT-solution with the resulting concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution
was thawed and diluted with assay medium directly before use.
- MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium), procured by Life Technologies GmbH,batch no.: 2125609.
- Assay Medium: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured by MatTek In Vitro Life Science Laboratories, batch no.: 062520MJC.
- Isopropanol: CH3-CH(OH)-CH3, for synthesis., ≥ 99.5 %, batch no.: 457264070, used as extracting solvent for formazan.
- DPBS-buffer: DPBS-buffer solution was used for the rinsing of the tissues, solvent for MTT concentrate and also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared in the testing facility. Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 042820MSA): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 L. Composition of the subset from LAUS GmbH (batch no.: T20190910): KCl 0.4 g, KH2PO4 0.4 g, NaCl 16.0 g, Na2HPO4 * 2H2O 2.87 g, H2O ad 2 L. The buffer which was procured by MatTek Corporation was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared by the testing facility was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.

PERFORMANCE OF THE STUDY
- Pre - Tests: It was tested whether the test item develops a colour without MTT addition. 25.1 mg test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. The resulting solution was colourless, therefore no binding capacity had to be tested. The test item was tested for the ability of direct MTT reduction. 26.3 mg test item were added to 1 mL of MTT solution and the mixture was
incubated in the dark at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. Untreated MTT medium was used as control. The MTT solution
did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.
- Pre-Incubation of Tissues: All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive
control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells.The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 1% CO2 and ≥ 95% relative humidity for 1 hour. After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity for 20 hours and 35 minutes.
- Treatment: One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to
ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used for treatment with the test item: replicate 1= 24.8 mg, replicate 2 = 26.4 mg, replicate 3 = 26.6 mg. The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size. Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).The surface of the inserts was then carefully dried with a sterile cotton tipped swab. Then, the tissues were set in the incubator for 22 hours and 50 minutes at 37 ± 1°Cand 5.0 ± 1% CO2 and ≥ 95% relative humidity.
- Medium Renewal: After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 20 hours for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
- MTT Assay: After a total incubation time of 42 hours and 50 minutes a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution (1 mg/ml) in
each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at
37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity. After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature. After 2 hours, the
inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was
thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well- plate which was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 µl isopropanol each, serving as blank.

Control samples:

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

Replicate 1 = 24.8 mg
Replicate 2 = 26.4
Replicate 3 = 26.6 mg

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours and 50 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

one

Value:

94.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD = 5.7%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the skin irritation study was demonstrated in a proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline)
were tested. All of the 10 proficiency chemicals were correctly categorized.Therefore, the proficiency of the skin irritation study was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD of negative control: 1.5
- Acceptance criteria met for positive control: yes. % tissue viability of positive control SDS: 3.9
- Acceptance criteria met for variability between replicate measurements: 4.6% (negative control), 0.0% (positive control), 5.7% (test item).
- Range of historical values if different from the ones specified in the test guideline: negative and positive controls are in the range of historical values.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified for skin irritation according to CLP criteria

Conclusions:

According to the in vitro results and OECD 439 criteria, the test item Titanium Glycolate Monoethanolamine is considered non irritant to skin.

Executive summary:

The in vitro study was performed in order to evaluate the potential of Titanium Glycolate Monoethanolamine to evoke skin irritation in a reconstructed huma epidermis (RhE) test method. The test method was performed according to OECD 439 and EU-Method B.46.

One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with the test item for 60 minutes.The test item was applied directly to each tissue and spread to match the tissue size cm2; (0.63 as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 = mean OD = 2.8, OD was 1.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.2% (required: . 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: = 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 94.3 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, the test item Titanium Glycolate Monoethanolamine is considered non- irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.