Trimethyl citrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

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:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes

Type of study:

activation of keratinocytes

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:

6.1 Test Item
Designation in Test Facility: 17100206G
Date of Receipt: 02. Oct. 2017
Condition at Receipt: Room temperature, in proper conditions
6.1.1 Specification
The following information concerning identity and composition of the test item was provided 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 information was provided by the sponsor as well:
CAS No. 1587-20-8
EINECS-No. 216-449-2
Molecular formula C9H14O7
Molecular weight 234.2 g/mol
Vapour pressure unknown
Stability H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Production date 01. Sep. 2017
6.1.2 Storage
The test item was stored in the test facility in a dry, closed vessel at room temperature (20 ± 5°C).
6.1.3 Preparation
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and culture medium. The test item was soluble in DMSO at the required concentration (200 mM). Therefore, DMSO was used as solvent.
Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM test item in DMSO was prepared. Subsequent dilution to 1% finally yielded a maximum concentration of 2000 µM in the test.
For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly pre-pared on the day of treatment.

In vitro test system

Details on the study design:

6.2.1 Negative Control
Name DL-Lactic acid
CAS no. 50-21-5
Solvent DMSO
Supplier: Sigma-Aldrich
Purity: 90.5 %
Lot no.: BCBP5043V
Expiry Date: 26. Jan. 2021
Final concentration: 5000 µM
The solution was freshly prepared on the day of the experiment.
6.2.2 Positive Control
Name EGDMA (Ethylene glycol dimethylacrylate)
CAS no. 97-90-5
Solvent DMSO
Supplier: Sigma-Aldrich
Purity: 98 %
Lot no.: SHBG0572V
Expiry Date: 27. Jan. 2021
Final concentration: 120 µM
The solution was freshly prepared on the day of the experiment.
6.2.3 Solvent Control
Name DMSO
CAS no. 67-68-5
Supplier: Carl Roth
Purity: 99.5 %
Lot no.: 187256959
Expiry Date: 04. Apr. 2020
Final concentration: 1 %

The LuSens cell line was specially designed for this test system by the BASF (Ludwigshafen, Germany). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Test cells of passage 6 were used. For both main experiments cells of passage 8 were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Results and discussion

Positive control results:

The positive control induced a clear effect with an induction value of 5.0 fold in comparison to the solvent control.

In vitro / in chemico

Results

Key result

Run / experiment:

other: In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation

Parameter:

other: Fold induction = [(Lsample - Lblank) / (Lsolvent - Lblank)]

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

In the following table 9 a the criteria for acceptability as well as the corresponding results in experiment I and II are given.
All validity criteria were met. Therefore, the study is valid.

Any other information on results incl. tables

Table9‑a      Acceptability of experiment I and II

Criteria	Found inexperiment I	Found inexperiment II
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.	Positive controlFold induction: 5.0 Relative viability: 102.9 %	Positive controlFold induction: 5.0 Relative viability: 101.2 %
The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.	Negative control: Fold induction: 1.0 Relative viability: 111.5 % Growth control: Fold induction: 1.0 Relative viability: 133.7 %	Negative control: Fold induction: 1.1 Relative viability: 113.6 % Growth control: Fold induction: 0.9 Relative viability: 138.2 %
The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.	7.74 %	9.91 %
At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.	12 concentrations are analysable	12 concentrations are analysable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No critical reduction of growth was observed in all tested test item concentrations.
In all tested concentrations of the test item no substantial and reproducible dose depend-ent increase of luciferase induction was measured.
In conclusion, it can be stated that under the experimental conditions of this study, the test item, Trimethyl citrate, was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).