1,4-bis(methoxymethyl)benzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2019-01-16 to 2019-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(methoxymethyl)benzene

EC Number:

229-828-2

EC Name:

1,4-bis(methoxymethyl)benzene

Cas Number:

6770-38-3

Molecular formula:

C10H14O2

IUPAC Name:

1,4-bis(methoxymethyl)benzene

Test material form:

liquid

Specific details on test material used for the study:

Batch No.: A7503
Purity: 99.89%

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

VEHICLE
-Vehicle: dimethyl sulfoxide (DMSO)
-Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (clear colorless solution).

Dose Formulation
-Preparation of Test Item Stock, Spiking and Working Solutions: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at
200 mM (clear colorless solution). 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 with exposure medium 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%).
-Preparation of the Positive Control: The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%).
-Preparation of the Solvent Control: The solvent control was 1% DMSO in exposure medium.

Test System
-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 KeratinoSensTM cell line).
- Rationale: 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 KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).
- Source: Givaudan (Duebendorf, Switserland).

Experimental 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. The passage number used was P+5 in experiment 1, P+7 in experiment 2 and P+9 in experiment 3.
-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. Initially, the results of the first experiment were unreliable (large variation in the viability of the triplicates) and therefore this experiment was repeated. In total 3 valid experiments were performed.
- Luciferase Activity Measurement: 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 5 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 and cells were incubated for 3 - 4 hours at 37°C ± 1.0°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.

Vehicle / solvent control:

DMSO

Positive control:

EGDMA (120 M) [442D]

Results and discussion

Positive control results:

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 (37.9 μM and 40.2 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.94-fold and 2.28-fold in experiment 1 and 2, respectively).

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

86 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximum luciferase activity induction (Imax) was 2.24-fold.

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

1 875 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximum luciferase activity induction (Imax) was 1.52-fold.

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

1 501 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximum luciferase activity induction (Imax) was 1.59-fold.

Other effects / acceptance of results:

OTHER EFFECTS:
-Precipitation: No precipitation was observed at the start and end of the incubation period in the 96-well plates for Experiment 1, 2 and 3.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control and negative (solvent) control: yes
-The test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

The test item showed no toxicity. No IC30 and IC50 value was measured at any of the test concentrations in all experiments. 

In the first experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 86 µM) was measured. The maximum luciferase activity induction (Imax) was 2.24-fold. The result of this individual experiment is positive.

In the second experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1875 µM) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

Since the first two experiments were not concordant a third experiment was performed. 

In the third experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1501 µM) was measured. The maximum luciferase activity induction (Imax) was 1.59-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

The test itemis classified as negativesince positive results (>1.5-fold induction) were observed at test concentrations > 1000 µM in two out of three experiments.

Applicant's summary and conclusion

Interpretation of results:

other: negative

Conclusions:

The test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

To evaluate the ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD guideline 442D.

The test item was dissolved in dimethyl sulfoxide at 200 mM. 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 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed.

All experiments passed the acceptance criteria,the test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity. No IC30 and IC50 value was measured at any of the test concentrations in all experiments. 

In the first experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 86 µM) was measured. The maximum luciferase activity induction (Imax) was 2.24-fold. The result of this individual experiment is positive.

In the second experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1875 µM) was measured. The maximum luciferase activity induction (Imax) was 1.52-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

Since the first two experiments were not concordant a third experiment was performed. 

In the third experiment, a biologically relevant induction of the luciferase activity (EC1.5 value of 1501 µM) was measured. The maximum luciferase activity induction (Imax) was 1.59-fold. Since the EC1.5 > 1000 µM the result of this individual experiment is negative.

The test itemis classified as negativesince positive results (>1.5-fold induction) were observed at test concentrations > 1000 µM in two out of three experiments.

In conclusion, the test item is classified as negative (no biologically relevantactivation of theantioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.