Carboxylic acids, di-, C5-9

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Aug - 06 Sep 2019

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:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted June 2018

Deviations:

yes

Remarks:

dose formulations were no solutions, therefore sterile filtration of dose formualtions before application was not possible

GLP compliance:

yes (incl. QA statement)

Remarks:

Secrétariat général du GIPC-DGE-SI, Ivry-sur-Seine CEDEX, France

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C5-9

EC Number:

701-329-9

Molecular formula:

C5di: C5H8O4 C6di: C6H10O4 C7di: C7H12O4 C8di: C8H14O4 C9di: C9H16O4

IUPAC Name:

Carboxylic acids, di-, C5-9

In vitro test system

Details on the study design:

TEST METHOD
This in vitro test uses human adherent HaCaT keratinocytes, an immortalized cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence.
Potential skin sensitizers are applied to the cells at 12 different concentrations for a period of 48 hours. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase. The luciferase reporter gene is under control of a single copy of the ARE-element of the human AKR1C2. The luciferase production is measured by flash luminescence.
In parallel, cytotoxicity is measured by MTT reduction and is taken into consideration in the interpretation of the sensitisation results. This evaluation is performed in at least two independent validated runs.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 5 in both experiments

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium No. 1: Dulbecco’s Modified Eagle Medium (DMEM) containing GlutaMAX™, 1000 mg/L D-glucose and Na-Pyruvate and supplemented with 9.1% fetal calf serum (FCS) and 500 µg/mL G-418 geneticin.
Maintanence medium No. 2: DMEM with 9.1% FCS without G-418 geneticin.
Treatment medium: DMEM with 1% FCS without G-418 geneticin.
Freezing medium: DMEM with 20% FCS and 10% DMSO

TEST CONCENTRATIONS
0.20; 0.39; 0.78; 1.56; 3.13; 6.25; 12.5; 25; 50; 100; 200 and 400 μg/mL in culture medium containing 1% DMSO

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%
Positive control
- Substance: cinnamic aldehyde
- Final concentrations: 4 - 64 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h
- Temperature (°C): 37
- CO2 (%): 5
- relative humdity (%): 90

NUMBER OF REPLICATES: duplicates each in two independent experiments for the test item and three replicates each in two independent experiments for the negative and positive control.

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm

DETERMINATION OF LUMINESCENCE
After incubation, the supernatants from the white assay plates were discarded. The cells were washed once with D-PBS. A volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 minutes (± 2 minutes) at room temperature under orbital shaking. The plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- A volume of 50 μL of the luciferase substrate was added to each well,
- 1 sec after this addition, the luciferase signal was integrated for 2 seconds Luminometer with injectors and optical density reader (Varioskan Flash).

Results and discussion

Positive control results:

All acceptance criteria were met in both validated runs with the exception of the criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8" which was not fulfilled in the second validated run (i.e. Imax of 8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.33

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.34

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

For both runs, the Imax values were < 1.5 (i.e. 1.33 and 1.34 in the first and second runs, respectively) and thus no EC1.5 was calculated. No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in both runs.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (17.94% in experiment 1; 15.87% in experiment 2). Therefore the negative control confirmed the validity of the study.
- Acceptance criteria met for positive control:
- Acceptance criteria met for variability between replicate measurements: The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 in experiment 1 (6.71). In experiment 2, the luciferase activity was slightly above 8 (8.15). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control in this run, this was considered not to have any impact on the validity of the results and the study was therefore considered as validated. The calculated EC1.5 was between 7 and 34 μM (9.38 μM in experiment 1; 6.42 μM in experiment 2).
Therefore the positive control confirmed the validity of the study.

Any other information on results incl. tables

Table 1. Evaluation of viability of culures treated with the test item

	Concentrations (µg/mL)
Carboxylic Acids, di-, C4 -11	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Viability (%) in Run 1	100	113	112	116	108	105	111	108	113	110	107	110
Viability (%) in Run 2	104	113	109	104	104	102	103	103	97	99	103	104
Mean viability (%)	102	113	111	110	106	104	107	106	105	104	105	107
Geometric Mean (%)	102	113	111	110	106	104	107	106	105	104	105	107
SD	3	0	2	8	3	2	6	4	12	7	3	5

Table 2. Gene induction values, mean and SD values obtained after treatment with the test item in each run

	Concentrations (µg/mL)
Carboxylic Acids, di-, C4-11	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Induction values in Run 1	1.0	1.1	1.2	1.1	1.1	1.1	1.2	1.2	1.3	1.1	1.2	1.3
Induction values in Run 2	1.1	1.0	1.1	1.1	1.1	1.0	1.3	1.2	1.1	1.3	1.0	1.1
Mean induction	1.1	1.1	1.2	1.1	1.1	1.0	1.3	1.2	1.2	1.2	1.1	1.2
SD	0.0	0.1	0.0	0.0	0.0	0.1	0.1	0.1	0.1	0.1	0.1	0.1

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event "activation of keratinocytes"

Conclusions:

There is regulatory acceptance in the EU for the application of the KeratinoSens™ test method to address key event 2, keratinocyte activation, in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test item did not have a keratinocyte activating potential. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test item and therefore further evaluation and/or data generation is required.