Tetramethylcyclotetrasiloxane substituted dipropylether bisphenol A

Endpoint summary

Description of key information

A non-animal test strategy was followed in order to assess skin sensitizing properties of the substance. All tests were performed according the relevant OECD guidelines and under GLP principles (Klimisch 1 studies).

- DPRA: the substance was found to be incompatible with this test system.

- hCLAT: The results indicate that the substance has skin sensitizing properties.

- ARE-Nrf2 Luciferase Test: the substance was predicted to be positive for skin sensitisation.

Based on the predictions generated from the rule based models Derek Nexus and the OECD Toolbox both constituents of the substance are predicted to be non-sensitisers. The predictions from the Leadscope statistical model were all considered out of domain.

 

In conclusion, the data indicate that the substance has skin sensitizing properties. However, as the non-animal testing strategy does not allow in a final conclusion on skin sensitizing properties (including potency), a follow-up animal study will be initiated (as a last resort).

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:

weight of evidence

Study period:

02-November-2021 to 19-November-2021 (experimental dates)

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:

June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Preliminary solubility data indicated that the test item was soluble in dimethyl sulfoxide (DMSO) up to at least 52.03 mg/mL, which was in excess of the target concentration of 40.00 mg/mL.
The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (40 mg/mL). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article in the range 0.02 to 40 mg/mL.
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.2 to 400 µg/mL.
Aliquots of 50 µL of each of the final concentrations were transferred to give three luciferase replicates on a white walled plate and a single viability replicate on a clear walled plate. After addition off the test item or control samples, each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37ºC, 5% CO2.
After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO2. The MTT medium was then removed and 200 µL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.
The following parameters were calculated:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test article and positive control;
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The acceptance criteria were met and the study was considered to be acceptable.
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2.
The EC1.5 value for the positive control was 11.16 and 16.03 µM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 µM were 7.31 and 6.13 in Experiments 1 and 2, respectively.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

2.94

Cell viability:

119.21%

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

3.48 µg/mL

Cell viability:

119.21%

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

4.17

Cell viability:

103.75%

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

0.8 µg/mL

Cell viability:

103.75%

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

No significant cytotoxic effects occurred at the lowest concentration leading to >1.5 fold luciferase induction as cell viability at the EC1.5 determining concentrations was 119.21% and 103.75% in Experiments 1 and 2, respectively.

The cell viability was above 70% at all concentrations in both experiments.

The IC30 and IC50 values could not be calculated for Experiments 1 and 2 as all viability results were above 70%.

Assay Acceptance
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2.
The EC1.5 value for the positive control was 11.16 and 16.03 µM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 µM were 7.31 and 6.13 in Experiments 1 and 2, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 12.67% and 16.71% in Experiments 1 and 2, respectively.

Interpretation of results:

study cannot be used for classification

Remarks:

The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Conclusions:

Based on the results of an ARE-Nrf2 Luciferase Test, performed according to OECD guideline 442D
and according to GLP principles, the test item was predicted to be positive for skin sensitisation.

Executive summary:

An ARE-Nrf2 Luciferase Test was performed according to OECD guideline 442D and according to GLP principles.

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (40 mg/mL). The final test concentrations ranged from 0.02 to 40 mg/mL. Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates. Luminescence was measured.

A dose response was observed in both experiments. The Imax was calculated to be 2.94 and 4.17 in Experiment 1 and Experiment 2 respectively. Cell Viability of 119.21% and 103.75% were observed in Experiment 1 and Experiment 2, respectively. The EC1.5 was calculated to be 3.48 μg/mL and 0.80 μg/mL for Experiment 1 and Experiment 2, respectively.

The results for the negative control (DMSO) confirmed the validity of the assay.

All four conditions required for a positive prediction were met in both experiments, therefore the test item was considered to be positive in the ARE-Nrf2 Luciferase Test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2020 to March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

* Dose Finding Assay:
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidiumiodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.

* CD86/CD54 Expression Measurement:
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on the same day provided that for each run:
a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and
b) Independently harvested cells were used (i.e. cells were collected from different culture flasks).

On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10E6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10E6 cells per well).

The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).

After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).

The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

For the positive control, RFI values were =150% for CD86 and =200% for CD54, and cell viability was >50% in each independent run.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

314.14 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

In both experiments, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations.
The EC150 value for CD86 calculated by linear regression of endpoint assay data was
314.14 µg/mL. No EC200 value was calculated for CD54 as this marker was negative in both experiments.

All assay acceptance criteria were met. The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run. In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions. For the positive control, RFI values were 150% for CD86 and 200% for CD54, and cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Dose Finding Assay: Cell Viability

Viability (%) at Concentration (μg/mL)
Run	7.81	15.63	31.25	62.5	125	250	500	1000
1	99.1	98.6	99	98.8	98.7	98.1	98.3	98.1
2	98.8	98.6	98.8	98.9	99	98.2	98.3	97.9

Expression Assay: MFI and Cell Viability Values

Experiment 1

Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI		Viability
	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
279.08	1425	575	481	944	94	97.5	96.6	97.0
334.90	1567	561	480	1087	81	96.4	96.0	96.2
401.88	1420	544	469	951	75	96.3	95.7	96.0
482.25	1224	557	463	761	94	97.7	96.5	95.6
578.70	1244	520	423	821	97	99.1	96.8	96.8
694.44	1453	573	485	968	88	96.7	95.1	95.4
833.33	1346	589	493	853	96	96.4	95.3	95.4
1000.00	1197	587	497	700	90	96.9	95.8	96.8
Culture medium	922	555	467	455	88	98.9	98.8	98.5
DMSO 0.2%	940	498	418	522	80	99.6	99.1	99.3
DNCB 4 μg/mL	2475	1687	580	1895	1107	84.9	80.9	82.3

Experiment 2

Concentration (µg/mL)	MFI (Geo Mean)			Corrected MFI		Viability
	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
279.08	1139	591	502	637	89	95.9	96.2	94.9
334.90	1350	568	471	879	97	95.6	95.1	94.8
401.88	1380	587	505	875	82	93.1	95.1	93.5
482.25	1254	568	489	765	79	94.1	94.5	93.8
578.70	1350	583	511	839	72	94.5	93.6	93.8
694.44	1354	603	529	825	74	92.6	93.3	92.5
833.33	1331	605	525	806	80	95.4	92.5	94.2
1000.00	1207	604	510	697	94	97.2	95.5	94.7
Culture medium	913	539	457	456	82	98.3	98.4	98.1
DMSO 0.2%	981	555	455	526	100	98.7	98.5	98.3
DNCB 4 μg/mL	2173	1115	569	1604	546	84.9	85.1	85.5

The relative fluorescence intensity (RFI) values:

Concentration (μg/mL)	RFI (CD86)		RFI (CD54)
	Exp1	Exp2	Exp1	Exp2
279.08	181	121	118	89
334.90	208	167	101	97
401.88	182	166	94	82
482.25	146	145	118	79
578.70	157	160	121	72
694.44	185	157	110	74
833.33	163	153	120	80
1000.00	134	133	113	94

Interpretation of results:

study cannot be used for classification

Remarks:

The data can only be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Conclusions:

The results of an in vitro skin sensitization test, performed according to OECD/EC guidelines and under GLP principles indicate that the test item has skin sensitizing properties.

Executive summary:

An in vitro skin sensitisation study (human Cell Line Activation Test) was conducted to investigate the potential of the test item to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1 by quantifying changes in the expression of cell surface markers (CD86 and CD54). For the dose finding assay, the test item was formulated in dimethyl sulphoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 μg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 279.08 to 1000 μg/mL (after dilution in medium) were used. Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10⁶ cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In the two experiments performed, the RFI values for CD86 were >150% (with cell viability >50%) at multiple concentrations and the RFI values for CD54 were <200% at all concentrations. The EC150 value for CD86 was calculated to be 314.14 μg/mL. All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

As a result, the substance was considered to be positive in the human Cell Line Activation Test.

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

In silico assessment

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

no guideline followed

Principles of method if other than guideline:

Predictions of skin sensitisation endpoints were made using the rule-based model Derek Nexus v6.1.0 and the statistical model Leadscope Model Applier v3.1.0-4. Chemical and biological reactivity profiles relevant for skin sensitisation were obtained using OECD (Q)SAR Toolbox v4.5.

The relevant QMRFs and report outputs are embedded in the report, which is attached.

The Derek alert is equivocal, meaning there is an equal weight of evidence for and against the proposition. The example training structure provided (1,4-bis(vinyloxy)methyl]cyclohexane) contains two vinyloxy fragments, whereas Constituent 1 has a methyl substituent in the α position which is likely to reduce its reactivity. The potential to form a Schiff base is also considered low for Constituent 1. Furthermore it was noted that there were no equivalent alerts raised in OECD Toolbox. The LLNA EC3 prediction was considered to be of low reliability due to the low number of nearest neighbours used in the prediction and low structural smilarity with Constituent 1. Therefore the alert and the LLNA EC3 prediction are refuted.
The Leadscope predictions were all considered out of domain since many fragments in the molecule were not included in the model training sets. The out of domain positive prediction for the h-CLAT Model was investigated and was considered of no relevance.

The in silico prognosis following expert evaluation is that an equivocal alert for skin sensitisation in a single model for Constituent 1 is refuted and therefore Constituent 1 is predicted to be a non-sensitiser.

The consistent in silico prognosis from the two alert based models (Derek and OECD Toolbox) is that
Constituent 2 is predicted to be a non-skin sensitiser. Since there were no misclassified or unclassified
features in the Derek prediction this is accepted.
The Leadscope predictions were all considered out of domain since many fragments in the molecule were not included in the model training sets. The out of domain positive prediction for the h-CLAT Model was investigated and was considered of no relevance.

The in silico prognosis following expert evaluation is that Constituent 2 is predicted to be a non-sensitiser.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the predictions generated from the rule based models Derek Nexus and the OECD Toolbox both constituents of the registered substance are predicted to be non-sensitisers. The predictions from the Leadscope statistical model were all considered out of domain.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available non-animal test data, it is concluded that there are indications that the registered substance has skin sensitizaing properties.

The substance is therefore classified Skin Sens. 1, according to Regulation (EC) No 1272/2008. It is of note that a follow-up animal study is planned to further determine the potency.