Reaction products of Soya and Linseed Oil Fatty Acids with Bisphenol A diglycidylether

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01May 2018 to 29June 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:

adopted February, 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

216-823-5

EC Name:

2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Cas Number:

1675-54-3

Molecular formula:

C21H24O4

IUPAC Name:

2,2'-[propane-2,2-diylbis(4,1-phenyleneoxymethylene)]dioxirane

Constituent 2

Reference substance name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

EC Number:

613-885-9

Cas Number:

66070-79-9

Molecular formula:

(C15 H16 O2 . C3 H5 Cl O . Unspecified . Unspecified)x

IUPAC Name:

Fatty acids, linseed-oil, polymers with bisphenol A, epichlorohydrin and soya fatty acids

Test material form:

liquid: viscous

Details on test material:

Clear colourless viscous liquid

In vitro test system

Details on the study design:

Test Chemical Preparation
No correction was made for the composition/purity of the test item. A solubility test was performed and 100 μg/mL concentration (10 mg/mL stock) was selected as highest concentration for the main assay (limit of solubility).

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 10 mg/mL (clear colorless solutions). 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 in the assay resulting in final test concentrations of 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098 and 0.049 μg/mL (final concentration DMSO of 1%). In the second and third experiment, final concentrations of 100, 67, 44, 30, 20, 13, 8.8, 5.9, 3.9, 2.6, 1.7 and 1.2 μg/mL (final concentration DMSO of 1%) were used (1.5-fold dilution series). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

Precipitation was observed at the start of the incubation period at concentrations of 50 μg/mL and upwards in the first experiment and 67 μg/mL and upwards in the second and third experiment. The test item precipitated at the highest dose level tested at the end of the incubation period.

Test item concentrations were used within 3.5 hours after preparation.


The Test System
The KeratinoSens™ cell line is a transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element. The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium. The KeratinoSens™ system is a validated in vitro skin sensitization test, which is recommended in international guidelines (e.g. OECD 442D).
ytotoxicity test

Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

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+4 in experiment 1, P+6 in experiment 2 and P+8 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±1.0oC in the presence of 5% CO2. 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 (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 KeratinoSensTM 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.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully
checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Results and discussion

Positive control 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 between 5 and 125 μM (109 μM, 23 μM and 50 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.11-fold, 4.20-fold and 4.53-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% (12%, 9.3% and 14% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

4.7 µg/mL

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Remarks:

Could not be determined

Value:

0

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Results 3

Key result

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

6.2 µg/mL

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

2 µg/mL

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 5

Key result

Run / experiment:

run/experiment 3

Parameter:

IC30 [442D]

Value:

4.8 µg/mL

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 6

Key result

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

1.3 µg/mL

Cell viability:

100%

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, the test item is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Executive summary:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens

assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch F288I1K351 of the test item was a clear colourless viscous liquid. The test item was

dissolved in dimethyl sulfoxide at 10 mg/mL. 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 0.049 – 100 μg/mL (2-fold dilution series) in the first experiment. The

highest test concentration was considered to be the limit of solubility. In the second and third

experiment, a more narrow dose-response analysis was performed using a lower dilution

factor of 1.5-fold to investigate a potential induction in experiment 1 in more detail. The test

item precipitated at the highest dose level tested. Three independent experiments were

performed.

All experiments 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 between 5 and 125 μM (109 μM, 23 μM and 50 μM

in experiment 1, 2 and 3, respectively). A dose response was observed and the induction

at 250 μM was higher than 2-fold (3.11-fold, 4.20-fold and 4.53-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% (12%, 9.3% and 14% in experiment 1 and 2,

respectively).

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

functioned properly.

The test item showed toxicity (IC30 values of 4.7 μg/mL, 6.2 μg/mL and 4.8 μg/mL in

experiment 1, 2 and 3, respectively, and IC50 values of 5.1 μg/mL, 6.9 μg/mL and 5.1 μg/mL

in experiment 1, 2 and 3, respectively). In the first experiment no biologically relevant

induction of the luciferase activity (no EC1.5 value could be calculated) was measured at any

of the test concentrations. The maximum luciferase activity induction (Imax) was 1.43-fold,

leading to an individual run conclusion of negative. In the second experiment a biologically

relevant, dose-related induction of the luciferase activity (EC1.5 value of 2.0 μg/mL) was

measured and a maximum luciferase activity induction (Imax) of 89.84-fold was measured.

Since the EC1.5 was below the IC30 this led to an individual run conclusion of positive. An

additional third experiment was conducted to provide a final conclusion. In the third

experiment a biologically relevant, dose-related induction of the luciferase activity (EC1.5

value of 1.3 μg/mL) was measured as well. The maximum luciferase activity induction (Imax)

was 39.10-fold. Since the EC1.5 was below the IC30 this led to an individual run conclusion of

positive. The test item is classified as positive in the KeratinoSensTM assay since positive

results (>1.5-fold induction) were observed in 2 out of 3 experiments at test concentrations <

200 μg/mL with a cell viability of >70% compared to the vehicle control.

In conclusion, 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 report.