Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

EC Number:

247-556-2

EC Name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

Cas Number:

26264-05-1

Molecular formula:

C18H30O3S.C3H9N

IUPAC Name:

sodium dodecylbenzenesulfonate

Test material form:

liquid

In vitro test system

Details on the study design:

Test background: The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address this second key event. Skin sensitizers have been reported to induce genes that are regulated by the antioxidant response element (ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes. In the KeratinoSensTM cell line activation of the Nrf2 pathway results in luciferase expression which can be quantified easily.

Test item preparation: No correction was made for the composition/purity of the test item. A solubility test was performed. The test item was suspended in DMSO to a final concentration of 200 mM (hazy suspension). The stock was sonicated (Time: 14 min; Temp.: 21 - 27 °C). The 100-fold dilution of the 200 mM DMSO stock formed a homogeneous solution (final concentration 2000 µM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline). In the main experiments the test item was suspended in dimethyl sulfoxide (DMSO) at 200 mM (hazy supsension). The stock was sonicated (Time: 7 min; Temp.: 25 - 29 °C in experiment 1; Time: 12 min; Temp.: 18 - 24 °C in experiment 2). 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 concentrations of the test item were tested in triplicate. 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 2.5 hours after preparation.

Test system: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan. 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell plating and treatment: For testing, cells were 80-90% confluent. 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+13 in experiment 2. 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 items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.

Measurement of luciferase activity: 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 TECAN Infinite® M200 Pro Plate Reader 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.

Data analysis: The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Data interpretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Results and discussion

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.21 and the EC1.5 89 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.83 and the EC1.5 72 µM.

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 (89 µM and 72 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.21-fold and 2.83-fold in experiment 1 and 2, respectively).

In vitro / in chemico

Results

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Two independent experiments were performed. The cells were in these experiments incubated with the test substance in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test substance showed toxicity. The calculated IC30 was 35 µM and the calculated IC50 was 43 µM.
• A dose related luminescence activity induction was observed after treatment with the test substance. The Imax was 2.17 and the EC1.5 21 µM. A significant increase was observed in the Student’s t test and therefore considered as a positive result.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test substance showed toxicity. The calculated IC30 was 43 µM and the calculated IC50 was 48 µM.
• A dose related luminescence activity induction was observed after treatment with the test substance. The Imax was 3.11 and the EC1.5 17 µM. The induction observed at 31 µM was statistically not significant (Student’s t test p = 0.136) due to one low luminescence reading (individual luminescence data of 25505, 2514 and 19516). The induction was considered biologically relevant since the effect was dose related, clearly above 1.5-fold induction and similar to the effect observed in experiment 1.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.83 and the EC1.5 72 µM.

Both tests passed the acceptance criteria:
The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (12% and 16% in experiment 1 and 2, respectively). The positive control substances also provided the expected results. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Overall: the test substance showed toxicity (IC30 values of 35 µM and 43 µM and IC50 values of 43 µM and 48 µM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 21 µM and 17 µM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.17-fold and 3.11-fold in experiment 1 and 2 respectively. The test substance is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 µM with a cell viability of >70% compared to the vehicle control.

Any other information on results incl. tables

Overview Luminescence Induction and Cell Viability of the test substance 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	0.94	1.08	1.07	1.03	1.20	2.17^***	0.00	0.00	0.00	0.00	0.00	0.00
Exp 1 viability (%)	105.0	112.2	109.3	106.2	122.6	80.8	0.2	0.5	0.1	0.2	0.4	0.2
Exp 2 luminescence	1.26	1.27	1.22	1.26	1.39	3.11	0.00	0.00	0.00	0.00	0.00	0.00
Exp 2 viability (%)	105.1	100.9	104.9	95.5	106.0	109.9	-0.3	-0.1	-0.1	-0.1	-0.1	-0.1

^***p<0.001 Student’s t test

       
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	1.23	1.19	1.31	1.38	1.62^**	2.21^***
Exp 1 viability (%)	113.8	120.6	120.0	134.3	133.2	145.0
Exp 2 luminescence	1.05	1.05	1.28	1.43	1.93^***	2.83^***
Exp 2 viability (%)	98.3	94.5	107.5	110.3	109.0	118.7

^**p<0.01 Student’s t test,^***p<0.001 Student’s t test

      
Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	21	2.17	35	43
Test item Experiment 2	17	3.11	43	48
Pos Control Experiment 1	89	2.21	NA	NA
Pos Control Experiment 2	72	2.83	NA	NA

NA = Not applicable

Applicant's summary and conclusion

Interpretation of results:

other: used a weight-of-evidence approach for classification and labelling

Conclusions:

In conclusion, the test substance is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.