Cyclooctane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-07-13 to 2021-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Test material

Test material information

Constituent 1

Reference substance name:

Cyclooctane

EC Number:

206-031-8

EC Name:

Cyclooctane

Cas Number:

292-64-8

Molecular formula:

C8H16

IUPAC Name:

cyclooctane

Test material form:

liquid

In vitro test system

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution and serial dilutions
On the day of the experiment (immediately before treatment) the test item was dissolved in DMSO to prepare a stock solution. The highest test concentration for the first dose finding assay was 2000 μM and 300 μM in the second dose finding assay. For the MTT test (dose finding assay) twelve concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest concentration.
- Preparation of the positive controls
Positive control: EGDMA (final concentration 120 μM), solved in treatment medium including 1 % (v/v) DMSO
- Preparation of the solvent, vehicle and negative controls
Medium control: Treatment medium
Solvent control for the positive control: DMSO, final concentration 1 % (v/v) in treatment medium
Negative control: Lactic acid (final concentration 5000 μM), solved in treatment medium including 1 % (v/v) DMSO

DOSE RANGE FINDING ASSAY:
- Highest concentration used: The highest test concentration for the first dose finding assay was 2000 μM and 300 μM in the second dose finding assay.
- Solubility in solvents: Test item was sufficiently soluble.
- Cytotoxicity assessment performed: yes, calculation of CV75 was done
- Final concentration range selected on basis of:
With reference to the CV75 parameter of the first dose finding assay the highest tested concentration in main experiment 1 was 187 μM. The highest tested concentration for the main experiment 4 and 5 was 183 μM without reference to the CV75 values of the dose finding assays.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: test item: 3 per concentration; solvent control: 24; positive control: 5; negative control: 6; treatment medium control: 12
- Number of repetitions: three main experiments (with valid results); In total 5 main experiments were conducted.
- Test chemical concentrations:
first main experiment: 75.2, 90.2, 108, 130, 156, 187 μM
main experiment 4 and 5: 35.5,42.6, 51.1, 61.3, 73.5, 88.3, 106, 127, 153, 183 μM

- Application procedure: 24 h ± 30 min after seeding of the cells, seeding medium were removed and 150 μL of treatment medium were distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells.
- Exposure time: 48 ± 1 h
- Study evaluation and decision criteria used:
The luciferase fold induction is calculated using the following formula (OECD 442D): Fold induction=(Lsample-Lblank)/(Lsolvent-Lblank)
where
Lsample is the luminescence reading in the sample well (samples: test item concentrations, medium, negative and positive control)
Lblank is the luminescence reading in the blank well without cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent control
The arithmetic mean of the luciferase fold induction was calculated for each sample.
- Description on study acceptance criteria:
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥2.5, and the positive control should have a relative cell viability ≥70 % as compared to the solvent control.
• The average luciferase activity induction obtained with the negative control, 5000 μM lactic acid, as well as the basal expression of untreated cells should be <1.5-fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20 % in each main experiment.
• At least three test item concentrations should have cell viability of at least 70 % relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability <70 %, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.
If a test item is tested with a concentration < 2000 μM (or 2000 μg/ mL for substances with no defined MW) and no luciferase induction as well as no cytotoxicity are observed, a second main experiment should be performed using e.g. a 1.44 serial dilution factor based on the CV75 (i.e. starting with 1.44 x CV75) instead of the 1.2 serial dilution factor. If in the second main experiment cytotoxicity and luciferase induction are still not observed, a third main experiment should be conducted with the maximum concentration of 2000 μM (or 2000 μg/mL for substances with undefined MW). This main experiment should then be confirmed by performing a fourth main experiment (OECD 442D).

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): The passage numbers of the used LuSens cells were 5 and 9 in the cytotoxicity tests and 7, 11 and 13 in the LuSens test for the main experiments 1, 4 and 5, respectively. Each treatment well of a 96 well microtiter plate was seeded with 100 μL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % CO2
- Washing conditions: At the end of the incubation period (48 ± 1 h), the treatment medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+.
- Precipitation noted: no

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: The absorption and luminescence measurement of the LuSens samples were conducted with a Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany. The experimental values were determined and calculated using the software MikroWin 2010, Version 5.19. Control tests were conducted as part of the experiments.
- Plate used: 96 well microtiter plate
- Measurement of the luciferase activity: The Steady-Glo® mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo® mix.
At the end of the incubation period (48 ± 1 h), the treatment medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 sec per well.

DATA EVALUATION
- Cytotoxicity assessment:
The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance. The relative absorbance (= cell viability) as compared to the solvent control is calculated using this formula: relative absorbance [%] = 100 × ((absorbance sample – absorbance blank)/(absorbance solvent control – absorbance blank)).
absorbance sample is the MTT-absorbance reading in the sample well
absorbance blank is the MTT-absorbance reading in the blank well (no cells and treatment)
absorbance solvent control is the average MTT-absorbance reading in the wells with cells and solvent control
The arithmetic mean was calculated for each sample.
The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), is calculated by using the following equation: CV75 = Conc.>75 – ((Conc. < 75) x (%>75 – 75)/(%>75 - %<75))
Conc. >75 = max. measured concentration with the % of solvent control >75 % ≡ a)
Conc. <75 = min. measured concentration with the % of solvent control <75 % ≡ b)
% >75 = relative absorbance at a) in %
% <75 = relative absorbance at b) in %
The values stated in the report are rounded values.
- Prediction model used:
If the luciferase induction is ≥1.5-fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥70%) and at least 3 tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442D).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Results and discussion

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.08; ME 4: 4.14; ME 5: 3.57). The positive control had a relative cell viability ≥70 % as compared to the solvent control (ME 1: 99.43 %; ME 4: 109.16 %; ME 5: 113.23 %).

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: luciferase induction

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The luciferase induction was not above or equal to 1.5-fold compared to the solvent control in all tested concentrations.

Results 2

Group:

test chemical

Run / experiment:

other: second cytotoxicity test

Parameter:

CV75 [442D and 442E]

Value:

47.7 µM

Results 3

Group:

test chemical

Run / experiment:

other: first cytotoxicity test

Parameter:

CV75 [442D and 442E]

Value:

156.1 µM

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.08; ME 4: 1.07; ME 5: 0.30 %), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 0.99; ME 4: 0.66; ME 5: 0.27).
- Acceptance criteria met for positive control: Yes. For details refer to “Positive control results”.
- Acceptance criteria met for variability between replicate measurements: Yes. The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 6.6 %; ME 4: 14.5 %; ME 5:10.4 %). At least three test item concentrations had a cell viability of at least 70 % relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70 %.

Any other information on results incl. tables

Please refer to attached document under "Overall remarks, attachments". 

Applicant's summary and conclusion

Interpretation of results:

other: The test item is negative for the second key event of the skin sensitisation AOP.

Conclusions:

The test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens; OECD TG 442D) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of the skin sensitization Adverse Outcome Pathway (AOP)) of the test item.

To determine the dose range of test item concentrations, two dose finding assays were performed. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 μM (threshold of cytotoxicity: <70 % cell viability) in the first cytotoxicity assay and at a concentration of 178 μM in the second cytotoxicity assay. The CV75 value of the first cytotoxicity test was calculated as 156.1 μM and 47.7 μM in the second one.

The test item was tested in 5 independent main experiments, of which 2 main experiments yielded in invalid results, since acceptance criteria regarding cytotoxicity was not met. Only the results of valid main experiments (main experiment 1, 4 and 5) are reported and the original numbering is used.

The following concentrations of the test item were tested in the first main experiment: 75.2, 90.2, 108, 130, 156, 187 μM.

After treatment with the test item for 48 ± 1 h in the main experiment 1 the luciferase induction is <1.5-fold compared to the solvent control in 5 tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met.

To characterize the test item concentrations in more detail, two additional main experiments (main experiment 4 and 5) were conducted with 10 test item concentrations. The following concentrations of the test item were tested: 35.5,42.6, 51.1, 61.3, 73.5, 88.3, 106, 127, 153, 183 μM.

After treatment with the test item for 48 ± 1 h the luciferase induction was not above or equal to 1.5-fold compared to the solvent control in all tested concentrations. Since the test item is considered negative, the acceptance criteria regarding testing at least one cytotoxic concentration is met.

The LuSens prediction for the test item is considered negative.

The acceptance criteria were met:

- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.08; ME 4: 4.14; ME 5: 3.57).

- The positive control had a relative cell viability ≥70 % as compared to the solvent control (ME 1: 99.43 %; ME 4: 109.16 %; ME 5: 113.23 %).

- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.08; ME 4: 1.07; ME 5: 0.30 %), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 0.99; ME 4: 0.66; ME 5: 0.27).

- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 6.6 %; ME 4: 14.5 %; ME 5:10.4 %).

- At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability <70 %.

In conclusion, the test item did not activate the LuSens cells up to a concentration of 187 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP.