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

Endpoint summary

Description of key information

sensitising

- KeratinoSens assay (FA-BADGE): >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

- LLNA (BADGE): EC3 = 5.7%

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
This read-across hypothesis corresponds to scenario 2 of the Read-Across Assessment Framework (RAAF), ECHA, March 2017 - different compounds have qualitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the source substance BADGE predicts the toxicological and ecotoxicological properties of the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
for details see Justification for read-across attached to iuclid section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
for details see Justification for read-across attached to iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
for details see Justification for read-across attached to iuclid section 13

4. DATA MATRIX
for details see Justification for read-across attached to iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

other: EC B.42 (Skin Sensitisation: Local Lymph Node Assay)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J mice were obtained from Harlan (Indianapolis, Indiana), and were approximately 9-12 weeks at study start.
Each animal was evaluated to determine the general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed up to six per cage in clear plastic cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were identified via subcutaneously implanted transponders that were correlated to unique alphanumeric identification numbers.

Animals were provided rodent diet in pelleted form. Feed and municipal water was provided ad libitum.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0.3%, 1%, 3%, 10% or 30%) or vehicle (AOO, acetone/olive oil)

No. of animals per dose:

6

Parameter:

SI

Value:

0.9

Test group / Remarks:

0.3% in 4:1 acetone:olive oil

Parameter:

SI

Value:

1.1

Test group / Remarks:

1% in 4:1 acetone:olive oil

Parameter:

SI

Value:

1.5

Test group / Remarks:

3% in 4:1 acetone:olive oil

Parameter:

SI

Value:

5.4

Test group / Remarks:

10% in 4:1 acetone:olive oil

Parameter:

SI

Value:

11.8

Test group / Remarks:

30% in 4:1 acetone:olive oil

Parameter:

EC3

Value:

5.7

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

A NOEL for the mouse LLNA was observed with a test concentration of 3% of the source substance BADGE. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of Soya/Linseed Oil Fatty Acid-BADGE reaction product was assessed by in vitro testing combined with read-across from the main constituent.

The in vitro testing battery is based on the skin sensitisation AOP (adverse outcome pathway). This AOP identifies four key events (KEs) with KE1, the covalent binding to skin proteins (termed haptenation) either of the parent substance or of its reactive derivatives following abiotic/metabolic activation, which is postulated to be the molecular initiating event (MIE), followed by KE2, the activation of epidermal keratinocytes, KE3, the activation (maturation) and mobilisation of Langerhans cell and dermal dendritic cells (DC), and KE4, the DC-mediated antigen presentation to naïve T-cells and proliferation/activation of allergen specific T-cells.

Soya/Linseed Oil Fatty Acid-BADGE reaction product is a UVCB substance; therefore, the DPRA is not applicable. Thus, no data are available for key event 1. A KeratinoSens Assay was conducted with FA-BADGE. In addition, a local lymph node assay is available, which was conducted with the source substance BADGE. A justification for read-across is attached to Iuclid section 13.

 

KeratinoSens Assay

The objective of this study was to evaluate the ability of Soya/Linseed Oil Fatty Acid-BADGE reaction product 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 (2015).

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.

 

Local lymph node assay

BADGE was examined in the local lymph node assay (LLNA). Groups of mice were treated with dose levels of 0.3, 1, 3, 10 and 30% BADGE in AOO (4:1 acetone:olive oil) for 3 consecutive days. On day 6, mice were injected with 3H-thymidine and 5 hours later incorporation of radiolabelled material in the auricular lymph nodes was determined.

A NOEL for the mouse LLNA was observed with a test concentration of 3% test item. The concentration that would cause a 3-fold increase in proliferation (EC3) was calculated to be 5.7% which is consistent with moderate dermal sensitization potential.

 

Conclusion

Overall, Soya/Linseed Oil Fatty Acid-BADGE reaction product is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

Based on reliable, adequate and relevant data, Soya/Linseed Oil Fatty Acid-BADGE reaction product is classified as skin sensitiser Category 1B according to regulation (EC) 1272/2008 and labelled with H317: May cause an allergic skin reaction and the signal word “Warning”.