1,1'-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June to 20 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSens assay, which is recommended in international guidelines (e.g. OECD 442D).

Test material

Test material information

1

Reference substance name:

1,1'-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane)

EC Number:

254-227-7

EC Name:

1,1'-(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane)

Cas Number:

38970-72-8

Molecular formula:

C18H34

IUPAC Name:

(4-cyclohexyl-4-methylpentan-2-yl)cyclohexane

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Batch: 170918339
Purity: 97.2%

In vitro test system

Details on the study design:

Plating of cells:
- 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.

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 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 ± 1 h at 37±1.0 °C in the presence of 5% CO2. In total 3 experiments were performed.

Luciferase Activity Measurement:
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) 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 KeratinoSens 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; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 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 (Sigma, Zwijndrecht, The Netherlands) 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 5 and 125 µM (49 µM, 77 µM and 83 µM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.41-fold, 2.12-fold and 2.68-fold in experiment 1, 2 and 3, respectively).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (µM)

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

Results 2

Run / experiment:

other: Experiment 2

Parameter:

other: EC 1.5 (µM)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

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. The Imax was 1.43 and therefore no EC1.5 could be calculated.

Results 3

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 (µM)

Value:

5.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 1.71 and the EC1.5 5.9 µM.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Three independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.004 – 7.8 µM (2-fold dilution steps) in experiment 1 and of 0.090 – 7.8 µM (1.5-fold dilution steps) in experiments 2 and 3 for 48 hours ± 1 h. 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.

One deviation was reported:

One of the luminescence readings for the DMSO vehicle control (9050) was excluded from the analysis of the positive control in experiment 1 as an outlier, since the variation was with 22.4% > 20%. And one of the luminescence readings for the EtOH vehicle control (2435) was excluded from the analysis of the test item in experiment 3, since the variation was with 20.2% > 20%. Based on evaluation of the data with Dixon’s Q-test these values were clear outliers.
Evaluation: After excluding this reading, still 17 vehicles were left for the calculations with a variation of 11% in experiment 1 and 3. Before excluding all other acceptation criteria were met. The exclusion was only affecting the positive control reading in experiment 1, which already met the acceptation criteria before exclusion. And in the third experiment had no influence on the positive result observed with the test item, since this was observed both before and after exclusion. Excluding these values has therefore no effect on the validity of the study.

This deviation was considered to not have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in the study.  However, due to coinciding increases in luciferase induction and cell viability the biological relevance can be questioned.