Trimethyl citrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Trimethyl citrate

EC Number:

216-449-2

EC Name:

Trimethyl citrate

Cas Number:

1587-20-8

Molecular formula:

C9H14O7

IUPAC Name:

1,2,3-trimethyl 2-hydroxypropane-1,2,3-tricarboxylate

Specific details on test material used for the study:

The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name Trimethyl citrate
Batch no. 20170901
Appearance white crystalline powder
Composition Trimethyl citrate
Purity 99.4%
Homogeneity homogeneous
Expiry date 01. Sep. 2019
Storage Room Temperature: (20 ± 5°C)

The following additional information, provided by the sponsor too, was relevant to the
conduct of the study, according to OECD 439:
CAS No. 1587-20-8
EINECS-No. 216-449-2
Stability H2O: unknown; EtOH: unknown; Acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: unknown; EtOH: unknown; Acetone: unknown; CH3CN: unknown; DMSO: unknown
Structural Formula not stated
SMILES Code not stated

Storage: The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).

In vitro test system

Test system:

other: EpiDermTM-Kit

Source species:

human

Cell type:

other: It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

Cell source:

other: It consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
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
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-218-SIT
Day of delivery: 16. Jan. 2018
Batch no.: 25874

Vehicle:

unchanged (no vehicle)

Details on test system:

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
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
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Brati-slava.
Designation of the kit: EPI-218-SIT
Day of delivery: 16. Jan. 2018
Batch no.: 25874

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amounts of Test Item
Tissue Amount
1 24.3 mg
2 25.6 mg
3 23.9 mg

Duration of treatment / exposure:

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 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

Duration of post-treatment incubation (if applicable):

After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Number of replicates:

3

Test animals

Species:

other: human

Details on test animals or test system and environmental conditions:

All working steps were performed under sterile conditions.
Incubator at 37 ± 1°C and 5 ± 0.5% CO2.

Test system

Details on study design:

7.1 Pre - Tests
7.1.1 Assessment of Coloured or Staining Test Items
It was tested whether the test item develops a colour without MTT addition. 25.8 mg test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.
7.1.2 Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability of direct MTT reduction. To test for this ability, 26.5 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 ± 0.5% CO2 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.
7.2 Pre-Incubation of Tissues
All working steps were performed under sterile conditions. For each treatment group (neg-ative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). 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 ± 0.5% CO2 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 ± 0.5% CO2 for 18 hours
55 minutes.

7.3 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:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spread-ing it to match the tissue size.

The following amounts were applied to the tissues:
Table 7.3 a Amounts of Test Item
Tissue Amount
1 24.3 mg
2 25.6 mg
3 23.9 mg

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 ± 0.5% CO2.
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, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
7.4 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 19 hours 5 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.
 

7.5 MTT Assay
After a total incubation time of 42 hours 40 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution 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 ± 0.5% CO2.
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 thor-oughly 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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 104.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item Trimethyl citrate is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was increased to 104.8% This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean value of relative tissue viability of the test item was increased to 104.8 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDerm^TMwere treated withTrimethyl citratefor 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; 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 values 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 1.9 % (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 increased to 104.8 %. 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,Trimethyl citrateis considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.