Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2018 - 29 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
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 sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

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)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The purpose of this study was to evaluate the ability of Polyambrol to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-6-propyl-2H-pyran-2-one
EC Number:
211-820-5
EC Name:
Tetrahydro-6-propyl-2H-pyran-2-one
Cas Number:
698-76-0
Molecular formula:
C8H14O2
IUPAC Name:
tetrahydro-6-propyl-2H-pyran-2-one
Test material form:
liquid
Details on test material:
Appearance: Clear colourless to pale yellow liquid
Storage: In refrigerator (2-8°C)
Specific details on test material used for the study:
Specific gravity: 0.998

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

CONTROLS:
Positive control: ethylene dimethacrylate glycol (purity: 98.3%), 7.8-250 µM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25). One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1 and P+8 in experiment 2.

TEST ITEM PREPARATION:
The test item was dissolved/suspended in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test item concentrations were used within 3.5 hours after preparation.

TEST CONCENTRATIONS:
- Both experiments: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM

MEDIA:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

TREATMENT OF CELLS:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours in a humid atmosphere of 80 - 100% (actual range 72 – 95 %) at 37.0 ± 1.0°C (actual range 35.6 – 37.2°C), in the presence of 5% ± 0.5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Results and discussion

Positive control results:
The EC1.5 of the positive control was between 26 and 125 μM (59 μM and 46 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (3.98-fold and 4.59-fold in experiment 1 and 2, respectively).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.12
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.
- In both experiments, the test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item in both experiments. The Imax were 1.17 and 1.12 in experiment 1 and experiment 2, respectively. From these Imax values, no EC1.5 could be calculated.


Both experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (26 μM and 46 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (3.98-fold and 4.59-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (12% and 10% in experiment 1 and 2, respectively).

Any other information on results incl. tables

Table 1 Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp 1 luminescence

1.13

1.00

1.06

1.14

1.17

1.14

1.12

1.15

1.09

0.95

0.90

0.80

Exp 1 viability (%)

98.9

106.6

99.5

96.6

98.2

91.1

93.2

89.3

95.2

101.8

103.7

95.7

Exp 2 luminescence

1.09

0.83

1.00

1.08

1.10

1.12

1.12

1.11

1.02

1.03

1.06

0.88

Exp 2 viability (%)

115.4

122.4

99.8

102.3

100.6

97.8

95.8

95.1

96.7

98.3

97.9

102.5

Table 2 Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.22

1.28

1.60***

1.86***

2.67***

3.98***

Exp 1 viability (%)

98.7

94.7

107.5

110.8

110.9

106.5

Exp 2 luminescence

1.03

1.13

1.31

1.72***

2.32***

4.59***

Exp 2 viability (%)

105.6

109.2

106.2

107.2

107.1

69.7

***p<0.001 Student’s t test

Table 3 Overview EC1.5, Imax, IC30 and IC50 Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.17

NA

NA

Test item Experiment 2

NA

1.12

NA

NA

Pos Control Experiment 1

26

3.98

NA

NA

Pos Control Experiment 2

46

4.59

NA

NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
This study is part of a weight of evidence approach on which the classification is based.
Conclusions:
The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations two independent experiments. The maximum luciferase activity induction (Imax) was 1.17-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens(TM) assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.
Based on the outcome of this KeratinoSens™ assay performed according to OECD guideline and GLP principles, Tetrahydro-6propyl-2H-pyran-2-one (delta octalactone) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.