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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10 Feb 2021 to 16 Apr 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

1
Chemical structure
Reference substance name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Molecular formula:
[(CnH2n+1)NH3+].[(CxH2x+1)-C6H4-S03-]
IUPAC Name:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl
Test material form:
liquid: viscous
Details on test material:
Chemical registery number : 953-378-2
Chemical name : Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

In vitro test system

Details of test system:
Keratinoses transgenic cell line [442D]
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in DMSO to a final concentration of 40 mg/mL (clear colourless solution). The stock solution was treated with ultrasonic waves until the test item had completely dissolved.
- Preparation of the test chemical serial dilutions: From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium.
- Preparation of the positive controls: A 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%).
- Preparation of the vehicle control: The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
- Stable dispersion obtained: YES

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of experiments: Three
- Test chemical concentrations: 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.10 and 0.05 µg/mL (final concentration DMSO of 1%) in the first experiment and in final test concentrations of 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.10, 0.05, 0.02 and 0.01 µg/mL (final concentration DMSO of 1%)
- Application procedure: The medium was removed and replaced with fresh exposure 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.
- Exposure time: 48 hours ± 1
- Study evaluation and decision criteria used: In accordance with the OECD TG 442D
- Description on study acceptance criteria: In accordance with the OECD TG 442D

SEEDING AND INCUBATION
- Seeding conditions: Distributed into 96-well plates (10,000 cells/well) in basic medium. Passage number used was P+4, P+10 and P+12 in experiment 1, 2 and 3, respectively
- Incubation conditions: Humid atmosphere of 80 - 100% (actual range 48 – 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 – 38.8°C)
- Precipitation noted: NO

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: TECAN Infinite® M200 Pro Plate Reader
- Lysate preparation: Steady-Glo Luciferase Assay Substrat

DATA EVALUATION
- Cytotoxicity assessment: The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability
Vehicle / solvent control:
DMSO
Negative control:
not applicable
Positive control:
EGDMA (120 M) [442D]

Results and discussion

Positive control results:
The EC1.5 of the positive control was within two standard deviations of the historical mean (35 µM, 45 µM and 27 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.65-fold, 5.08-fold and 3.59-fold in experiment 1, 2 and 3, respectively).

In vitro / in chemico

Resultsopen allclose all
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
IC30 [442D]
Value:
2 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC30 [442D]
Value:
2.2 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
IC30 [442D]
Value:
4.3 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
IC50 [442D]
Value:
2.5 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC50 [442D]
Value:
3.7 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
IC50 [442D]
Value:
6.6 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Value:
1.92 %
Cell viability:
89%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Value:
1.1 %
Cell viability:
96%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Group:
test chemical
Run / experiment:
run/experiment 3
Parameter:
Imax [442D]
Value:
1.22 %
Cell viability:
96%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Outcome of the prediction model:
negative [in vitro/in chemico]
Other effects / acceptance of results:
All tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (35 µM, 45 µM and 27 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.65-fold, 5.08-fold and 3.59-fold in experiment 1, 2 and 3, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.3%, 8.2% and 11% in experiment 1, 2 and 3, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Table 1          
Overview Luminescence Induction and Cell Viability of the test item in Experiment 1, 2 and 3






























































































































Concentration (µg/mL)



0.05



0.10



0.20



0.39



0.78



1.6



3.1



6.3



13



25



50



100



Exp 1 luminescence



1.85***



1.86***



1.92***



1.70***



1.65***



1.45



1.04



0.11



0.00



0.00



0.01



0.00



Exp 1 viability (%)



92



90



89



88



88



86



25



0.14



-0.15



-0.06



-0.08



-0.07



Concentration (µg/mL)



0.01



0.02



0.05



0.10



0.20



0.39



0.78



1.6



3.1



6.3



13



25



Exp 2 luminescence



0.93



0.94



0.97



1.07



1.05



1.10



0.96



0.96



0.73



0.29



0.00



0.00



Exp 2 viability (%)



124



103



103



98



95



96



87



76



60



7.2



0.05



0.11



Exp 3 luminescence



0.98



1.05



1.11



1.18



1.16



1.22



1.09



0.99



0.84



0.57



0.36



0.00



Exp 3 viability (%)



92



91



91



92



90



96



89



88



81



52



11



0.47



*** p<0.001 Student’s t test


 


Table 2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1, 2 and 3





































































Concentration (µM)



7.8



16



31



63



125



250



Exp 1 luminescence



1.08



1.30



1.47



1.77***



2.10***



2.65***



Exp 1 viability (%)



107



110



108



114



117



111



Exp 2 luminescence



1.05



1.05



1.17



1.93



1.93*



5.08



Exp 2 viability (%)



128



127



122



117



113



106



Exp 3 luminescence



1.07



1.33



1.57***



2.02***



2.77***



3.59***



Exp 3 viability (%)



99



96



99



110



107



109



*** p<0.001 Student’s t test, * p<0.05 Student’s t test


 


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






























































 



EC1.5 (µg/mL)



Imax



IC30 (µg/mL)



IC50 (µg/mL)



Test item Experiment 1



0.03



1.92



2.0



2.5



Test item Experiment 2



NA



1.10



2.2



3.7



Test item Experiment 3



NA



1.22



4.3



6.6



 



EC1.5 (µM)



Imax



IC30 (µM)



IC50 (µM)



Pos Control Experiment 1



35



2.65



NA



NA



Pos Control Experiment 2



45



5.08



NA



NA



Pos Control Experiment 3



27



3.59



NA



NA



NA = Not applicable


 


Table 4          
Historical Control Data for the KeratinoSens Studies



































 



Positive control



 



EC1.5 (µM)



Imax



Range


(mean ± 2x SD)



-3.0 – 120



-5.65 – 12.15



Mean



58.5



3.25



SD



30.7



4.45



n



503



503



SD = Standard deviation


n = Number of observations


The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2017 to November 2020.


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl (EC 953-378-2) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.
Executive summary:

The ability of Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl (EC 953-378-2) to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay was investigated.


The test item was dissolved in dimethyl sulfoxide (DMSO) at 10 in the first experiment and 2.5 mg/mL in the second and third experiment. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.05 – 100 µg/mL (2-fold dilution series) in the first experiment and in test concentrations of 0.01 – 25 µg/mL (2-fold dilution series) in the second and third experiment. The highest test concentration was considered to be the limit of solubility. No precipitate was observed at any dose level tested. Three independent experiments were performed.


All tests passed the acceptance criteria:



  • The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

  • The EC5 of the positive control was within two standard deviations of the historical mean (35 µM, 45 µM and 27 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.65-fold, 5.08-fold and 3.59-fold in experiment 1, 2 and 3, respectively).

  • Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.3%, 8.2% and 11% in experiment 1, 2 and 3, respectively).


Overall it is concluded that the test conditions were adequate and that the test system functioned properly.


In the first experiment, the test item showed toxicity (IC30 value of 2.0 µg/mL and IC50 value of 2.5 µg/mL). An induction of the luciferase activity (EC1.5 values of 0.03 µg/mL) was measured. The maximum luciferase activity induction (Imax) was 1.92-fold leading to an individual run conclusion of positive since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of >70% compared to the vehicle control.


In the second experiment, the test item showed toxicity (IC30 value of 2.2 µg/mL and IC50 value of 3.7 µg/mL). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations The maximum luciferase activity induction (Imax) was 1.10-fold leading to an individual run conclusion of negative since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/mL with cell viability < 70% compared to the vehicle control.


Since the first two experiments were not concordant, a third experiment was performed to provide a final conclusion. 


In the third experiment, the test item showed toxicity (IC30 value of 4.3 µg/mL and IC50 value of 6.6 µg/mL). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.22-fold leading to an individual run conclusion of negative since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/mL with cell viability
< 70% compared to the vehicle control.


Overall, the test item is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed in two out of three experiments at test concentrations < 200 µg/mL with cell viability < 70% compared to the vehicle control.


In conclusion, Benzenesulfonic acid, 4-C10-13-alkyl derivs.-, salts with amines, C16-C18 (even numbered) alkyl (EC 953-378-2) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.