reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2017 - 10 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

yes

Remarks:

Except that no solubility test was performed since the test item was already formulated in water. This deviation to the guideline was considered not to have any impact on the integrity of the study once the test item was soluble in a recommended vehicle.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

In vitro test system

Details on the study design:

Vehicle: water for injections
Negative control: DMSO; this negative control was applied to cells at a concentration of 1% in culture medium.
Positive control item: Cinnamic Aldehyde (CA). For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of two, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Criteria: interpretation of results
Acceptance criteria
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between two and eight. If the latter criterion was not fulfilled, the dose response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
- the average Coefficient of Variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean (first run)

Parameter:

other: EC1.5

Remarks:

expressed in µM

Value:

4.27

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Run / experiment:

other: mean (second run)

Parameter:

other: EC1.5

Remarks:

expressed in µM

Value:

13.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Run / experiment:

other: first run

Parameter:

other: Imax

Value:

2.16

Results 4

Run / experiment:

other: Second run

Parameter:

other: Imax

Value:

3.97

Other effects / acceptance of results:

First and second runs
All acceptance criteria were fulfilled for the positive and negative controls. The runs were therefore considered to be valid.
The results met the criteria of a positive response in both runs.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The two major constituents of the test item (dodecylbenzenesulfonic acid/CAS 27176-87-0 and cyclohexanamine/CAS 108-91-8) are classified H314 “causes severe skin burns and eye damage” according to the classification provided by companies to ECHA in REACH registrations

Conclusions:

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor, though with caution due to the H314 classification ("causes severe skin burns and eye damage") of the two major constituents of the test item.

Executive summary:

The objective of this study was to evaluate the potential of the test item to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

The study was performed according to the international OECD guideline No.442D (except that no solubility test was performed since the test item was already formulated in water) and in compliance with the principles of Good Laboratory Practice.

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte 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. 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.

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed.

Results

First run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered as validated.

 

This run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

.            no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period at any tested concentrations,

.            a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations = 15.63 µM,

.            the corresponding IC₃₀ and IC₅₀ were calculated to be 11.58 and 12.73 µM, respectively,

.            a statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at 7.81 µM with an apparent overall dose-response relationship up to the cytotoxic concentrations,

.            the I_max was 2.16 and the calculated EC_1.5 was 4.27 µM.

Thus, the results met the criteria of a positive response

Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

 

This run was performed using the following concentrations 1.43, 2.01, 2.84, 4.00, 5.64, 7.95, 11.2, 15.8, 22.3, 31.4, 44.3 and 62.5 µM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

.            no precipitate/emulsion was observed in any wells at the end of the 48-hour treatment period at any tested concentrations,

.            a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations = 31.4 µM,

.            the corresponding IC₃₀ and IC₅₀ were calculated to be 26.99 and 28.31 µM, respectively,

.            statistically significant gene-fold inductions above the threshold of 1.5 were noted in comparison to the negative control at 15.8 and 22.3 µM with an apparent overall dose-response relationship up to the cytotoxic concentrations,

.            the I_max was 3.97 and the calculated EC_1.5 was 13.72 µM.

Thus, the results met the criteria of a positive response.

 

Global analysis from both runs:

The geometric means IC₃₀and IC₅₀of the twovalidated runs were 17.68 and 18.98 µM, respectively.

 

The evaluation criteria for a positive response were met in both runs, the final outcome is therefore positive. This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Discussion

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay. However, it is worthy to note that the two major constituents of the test item are classified H314 "causes severe skin burns and eye damage" according to the classification provided by companies to ECHA inREACH registrations (information specified by the Sponsor in an email dated 31 March 2017 archived with the study files).

According to the EURL ECVAM Recommendation on the KeratinoSens assay for skin sensitization testing (European Commission - February 2014 (JRC 87551)), reactive chemicals that cause dermal corrosion/irritation without, however, being skin sensitizers, may lead to false positive results in the KeratinoSens test method.

Thus, in the context of an integrated approach to testing and assessment, this information should be considered and the result of this test be taken with caution.

Conclusion

Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor, though with caution due to the H314 classification (“causes severe skin burns and eye damage”) of the two major constituents of the test item.