ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol

Endpoint summary

Description of key information

The skin sensitisation potential of ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol has been assessed using (Q)SAR and investigated in two in vitro skin sensitisation studies as well as one in vivo study. The (Q)SAR assessment concluded that the substance is unlikely to be a skin sensitiser, but this could not be completely excluded due to the potential formation of dermal metabolites with sensitising potential.

Ambiguous results have been obtained in skin sensitisation studies conducted in vitro. In an in vitro study conducted according to OECD Test Guideline 442E, the substance gave negative results in the human cell line activation test and was not considered to have any sensitising potential. However, in an in vitro test conducted according to OECD 442D, the substance gave positive results in the LuSens assay and was considered to have skin sensitising potential. The resason not to perform the in chemico study accoding to OECD Test Guideline 422C, is that the substance is a UVCB substance. According to the test guideline on the In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), the test method should not be used for this type of substances and the test is therefore waived.

To further investigate the skin sensitisation potential of the substance, an in vivo test according to OECD Test Guideline 429 Skin sensitization: Local Lymph Node Assay was conducted. In the performed in vivo study, the substance was not considered as a skin sensitizer.

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:

15-26 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

29 July 2016

Deviations:

no

Principles of method if other than guideline:

OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) (adopted 29. July 2016)

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

Pre-test
A preliminary cytotoxicity test was performed to determine the concentrations to be used for the CD86/CD54 expression measurement in the main experiments. The following 8 concentrations of the test item were tested with an incubation time of 24 h in the pre-test: 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.3 µg/mL, 15.6 µg/mL and 7.8 µg/mL. 1.6*10e5 cells per well of a 96-well plate were exposed to each concentration of the test item for 24 hours at 37°C and 5% CO2. Following treatment, the cells were transferred into sample tubes and centrifuged for 5 minutes at 250*g. The supernatant was removed, the cell pellet was suspended in 600 µL staining buffer and 200 µL samples of the cell suspension were transferred into one of the wells of a 96-well round-bottom plate. The cells were washed three times with 200 µL staining buffer. Cells were resuspended in 150 µL staining buffer and 7.5 µL PI solution (12.5 µg/mL) was added to achieve a final concentration of 0.595595 µg/mL. The plate was incubated for 15 min on ice and 5 minutes at room temperature in the dark. Afterwards, the PI uptake was analysed by flow cytometry. In total 10000 viable cells (PI negative) were acquired. Cell viability was automatically calculated by the flow cytometer.

Main test
The performance of experiments I, II and III was identical: eight test item concentrations were tested in each experiment. 1 *10e6 cells per well of a 24-well plate were exposed to each concentration of the test item for 24 hours at 37°C and 5.0% CO2. During treatment the plates were closed with tape to avoid cross-contamination. Following treatment, the solutions were transferred into sample tubes and washed twice with 1 mL staining buffer and centrifuged (250 *g for 5 minutes). Cells were resuspended in 600 µL blocking solution and incubated on ice for 15 minutes. 180 µL cell suspension were added to three wells of a 96-well round bottom plate. Plates were centrifuged at 250 *g for 5 minutes and the supernatant discarded. 50 µL of the three FITC-labelled antibody solutions were added to one of the three wells and the plate incubated on ice for 30 minutes in the dark. Before use, the antibodies were diluted 3:25 v/v for CD86, 3:50 v/v, for CD54 and IgG1, with staining buffer. Following incubation on ice, the cells were washed 3 times with 200 µL staining buffer and centrifuged (250 *g for 5 minutes) before 150 µl staining buffer and 7.5 µL PI working solution were added to each well. The plate was incubated for 15 minutes on ice and 5 minutes at room temperature in the dark before measurement using a flow cytometer with FITC detectio (excitation: 488 nm. 10000 viable cells (PI negative) were acquired and analysed.

Viabilty was directly measured and calculated by the flow cytometer and is reported as % values. For evaluation of CD54/CD86 expression, the mean fluorescence intensity (MFI) was also analysed uisng the flow cytometer software. The EC200 value for CD54 was also calculated.

Positive control results:

Prior to routine use, the validity of the h-CLAT test at the testing facility was demonstrated in a proficiency study. In this study, 10 proficiency chemicals indicated by the OECD 442E guideline were tested. As prescribed by the guideline, more than 7 of the results were correctly categorized. Therefore, the proficiency of the test system was demonstrated. For all control substances historical data are available which demonstrate the reliability and the validity of those substances. Prior to use in the pre-test and the experiment, the proficiency of the cells was demonstrated in a reactivity check. Only those cells which passed the reactivity check were used for the pre-test and the main experiments. The runs for Experiment I and II were performed on the same day, provided that for each run: a) independent fresh stock solutions and working solutions of the test item and antibody solutions were prepared and b) independently harvested cells were used (cells came from the same passage and were collected from different culture flasks.)

Key result

Run / experiment:

other: Experiments I, II, III

Parameter:

other: RFI value

Remarks:

CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment I, III

Parameter:

other: RFI value

Remarks:

CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment II

Parameter:

other: RFI value

Remarks:

CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

the RFI of CD54 was >200% at one concentration (1000 µg/mL); therefore a third repetition was performed.

Other effects / acceptance of results:

The assay is considered acceptable if it meets the following criteria:
The cell viabilities of medium and solvent/vehicle controls are higher than 90%.
In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive crite-ria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
In the positive control, RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.
For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.

If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.

Negative results are acceptable only for test items exhibiting a cell viability of less than 90 % at the highest concentration tested. If the cell viability at 1.2 × CV75 is equal or above 90 % the negative result should be discarded. In such a case another Pre-test has to be performed to refine the dose selection. It should be noted that when 5000 µg/mL in PBS or medium, 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item, a negative result is acceptable even if the cell viability is above 90 %.

All validity criteria were met. Therefore, the study was considered to be valid.

Results of Experiment I

	Concentration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]
Medium	-	97.07%	CD86	1289	179
		97.17%	CD54	1000	139
		97.35%	ISO	722
DMSO	-	97.89%	CD86	1243	168	89
		97.47%	CD54	1017	137	100
		97.81%	ISO	740
DMSO	-	98.01%	CD86	1280	170	93
		98.37%	CD54	1013	134	93
		97.87%	ISO	754
DNCB	4.0	82.35%	CD86	4886		756
		83.40%	CD54	1785		339
		81.70%	ISO	907
Test Item	279.1	97.39%	CD86	1261		96
		97.49%	CD54	1050		99
		97.13%	ISO	777
Test Item	334.9	97.20%	CD86	1238		92
		96.99%	CD54	1044		98
		96.94%	ISO	773
Test Item	401.9	96.53%	CD86	1267		93
		96.89%	CD54	1066		96
		96.81%	ISO	799
Test Item	482.3	94.93%	CD86	1470		125
		95.47%	CD54	1142		109
		95.36%	ISO	840
Test Item	578.7	94.61%	CD86	1457		129
		90.74%	CD54	970		59
		90.67%	ISO	807
Test Item	694.4	94.34%	CD86	1595		142
		94.53%	CD54	1184		109
		94.22%	ISO	883
Test Item	833.3	92.55%	CD86	1612		146
		93.80%	CD54	1202		117
		93.08%	ISO	878
Test Item	1000.0	96.00%	CD86	1354		98
		95.11%	CD54	1102		86
		95.51%	ISO	863

Results of Experiment II

	Concentration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]
Medium	-	98.05%	CD86	1357	195
		98.06%	CD54	970	139
		97.84%	ISO	697
DMSO	-	97.66%	CD86	1159	169	72
		98.30%	CD54	965	141	103
		97.21%	ISO	685
DMSO	-	98.22%	CD86	1168	166	70
		98.48%	CD54	968	138	97
		98.21%	ISO	704
DNCB	4.0	83.00%	CD86	4856		861
		82.56%	CD54	1748		336
		83.80%	ISO	861
Test Item	279.1	97.44%	CD86	1338		125
		97.47%	CD54	1059		112
		97.27%	ISO	745
Test Item	334.9	96.50%	CD86	1288		111
		97.24%	CD54	1055		104
		97.00%	ISO	763
Test Item	401.9	96.30%	CD86	1303		111
		96.17%	CD54	1065		104
		96.49%	ISO	775
Test Item	482.3	97.71%	CD86	1329		120
		97.68%	CD54	1107		124
		97.66%	ISO	759
Test Item	578.7	91.79%	CD86	1358		111
		93.07%	CD54	1145		113
		93.39%	ISO	830
Test Item	694.4	95.68%	CD86	1319		111
		95.84%	CD54	1123		118
		94.93%	ISO	793
Test Item	833.3	94.16%	CD86	1560		141
		93.72%	CD54	1165		98
		94.97%	ISO	892
Test Item	1000.0	91.51%	CD86	1321		149
		94.61%	CD54	1203		210
		91.34%	ISO	616

Results of Experiment III

	Concentration	Viability	Antibodies	MFI Value	MFI ratioto Isotype	RFI Value
	[µg/mL]				[%]
Medium	-	97.56%	CD86	1327	195
		97.75%	CD54	1003	148
		97.66%	ISO	679
DMSO	-	97.91%	CD86	1182	175	78
		97.52%	CD54	986	146	96
		97.11%	ISO	675
DMSO	-	97.66%	CD86	1198	176	80
		97.74%	CD54	1017	149	103
		97.46%	ISO	682
DNCB	4.0	86.51%	CD86	4781		760
		86.57%	CD54	1800		281
		85.06%	ISO	859
Test Item	279.1	97.28%	CD86	1268		106
		96.90%	CD54	1052		103
		96.97%	ISO	733
Test Item	334.9	96.99%	CD86	1245		102
		97.06%	CD54	1051		103
		96.57%	ISO	730
Test Item	401.9	95.43%	CD86	1353		119
		96.02%	CD54	1076		105
		96.51%	ISO	751
Test Item	482.3	94.10%	CD86	1396		124
		94.44%	CD54	1130		116
		95.22%	ISO	768
Test Item	578.7	95.15%	CD86	1333		102
		95.55%	CD54	1142		105
		95.84%	ISO	817
Test Item	694.4	92.09%	CD86	1217		76
		95.24%	CD54	1158		105
		94.87%	ISO	832
Test Item	833.3	92.37%	CD86	1532		132
		91.56%	CD54	1186		104
		91.18%	ISO	862
Test Item	1000.0	91.01%	CD86	1313		105
		90.81%	CD54	950		54
		92.32%	ISO	783

Interpretation of results:

GHS criteria not met

Conclusions:

For CD86/CD54 expression measurement, the test item is tested in at least two independent runs to derive a single prediction. The h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:
− The RFI of CD86 is ≥ than 150 % at any tested concentration with cell viability ≥ 50 %
− The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %
If the first two runs are concordant for both markers, a third experiment is not necessary. Then, the study is completed. If the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered positive.

In accordance to these classification criteria the test item ε-Caprolactone, oligometric reaction products with 2,2'-oxydiethanol was negative in the h-CLAT and is therefore considered not having the potential to activate dendritic cells and therefore to be a non-sensitizer.

Executive summary:

This in vitro study was performed to assess the sensitising potential of the test item ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, by quantifying changes in the expression levels of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. Three valid experiments with a treatment period of 24 hours were performed. In the experiments, the highest concentration (1000 µg/mL) was chosen based on cytotoxicity results obtained in the pre-test. A geometric series of seven dilutions was prepared. DMSO was used as solvent at a final concentration of 0.2% in the culture medium. 2,4-dinitrochlorobenzene (DNCB) was used as the positive control. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable. 

No cytotoxic effect was detected in any experiment at any of the test concentrations. In Experiments I and II, the RFI of CD86 was <150%. In Experiment I the RFI of CD54 was <200%. In Experiment II, the RFI of CD54 was>200% at the highest test item concentration (1000 µg/mL). Therefore a third repetition was performed. In Experiment III the RFI of CD86 was <150% and the RFI of CD54 was <200%. As the majority of the results of the three individual runs is negative, the test item is considered as negative. All acceptance criteria were met and therefore, the study was considered valid.

It can be concluded that, under the experimental conditions of this study, the test item, ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol was negative in the h-CLAT and is therefore considered as not having the potential to activate dendritic cells and is a non-sensitiser.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

(Q)SAR assesment

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

OECD QSAR Toolbox was used to identify structural alerts for ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol relating to skin sensitisation. ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol is an oligomeric substance; modelling was therefore performed using a representative structure (DEG-2ECL). Relevant models included in the Toolbox are: DPRA cysteine peptide depletion, DPRA lysine peptide depletion, Protein binding (OASIS v1.4), Protein binding (OECD), Protein binding potency, Keratinocyte gene expression, Sensitisation alert (OASIS v1.4). OECD QSAR Toolbox was further used to predict the potential skin metabolites of CAPA ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol; the four skin metabolites were similarly screened for structural alerts relating to skin sensitisation.

The outcome of the QSAR assessment can be used as part of a weight of evidence assessment to address the hazard identification and classification for this particular endpoint.

Qualifier:

no guideline followed

Principles of method if other than guideline:

(Q)SAR assessment of skin sensitisation potential using OECD Toolbox

GLP compliance:

no

Remarks:

Not applicable

Key result

Parameter:

other: Skin sensitisatioj potential

Remarks on result:

other:  The potential for skin sensitisation does not appear to be very high, but cannot be completely discounted.

Structural alerts for ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol and its predicted skin metabolites

Substance	ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol	Metabolite 1	Metabolite 2	Metabolite 3	Metabolite 4
DPRA cysteine peptide depletion	No alert	Low reactive: long-chain aliphatic aldehyde	No alert	No alert	No alert
DPRA lysine peptide depletion	No alert	No alert	No alert	No alert	No alert
Protein binding (OASIS v1.4)	No alert	Schiff base formation: monocarbonyl	No alert	No alert	No alert
Protein binding (OECD)	No alert	Schiff base formation: monocarbonyl	No alert	No alert	No alert
Protein binding potency	No alert	Not possible to classify	No alert	No alert	No alert
Keratinocyte gene expression	No alert	Moderate gene expression: aldehyde	No alert	No alert	No alert
Sensitisation alert (OASIS v1.4)	No alert	Schiff base formation: carbonyl	No alert	No alert	No alert

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the assessment, it is concluded that ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol is unlikely to be a skin sensitiser; however this cannot be completely excluded due to the potential formation of dermal metabolites with sensitising potential.

Executive summary:

OECD QSAR Toolbox was used to identify structural alerts for ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol relating to skin sensitisation.  ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol is an oligomeric substance; modelling was therefore performed using a representative structure (DEG-2ECL). The representative structure of ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol does not show any structural alerts relating to skin sensitisation. OECD QSAR Toolbox was further used to predict the potential skin metabolites of ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol; the four skin metabolites were similarly screened for structural alerts relating to skin sensitisation. Three metabolites do not show any alerts; however one metabolite (Metabolite 1) shows a number of alerts due to the presence of an aldehyde (carbonyl) group. The extent of the dermal metabolism of ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol is unknown; however viable skin does possess esterase activity. The metabolite may therefore be produced in studies in vivo or in vitro using metabolically competent skin cells. The skin sensitisation potency of Metabolite 1 is unknown, but this is ranked as low potency (DPRA cysteine peptide depletion) or moderate potency (keratinocyte gene expression). The potential for a positive result in any assay will be determined both by the extent of metabolism and the potency of the metabolite. The potential for skin sensitisation does not appear to be very high, but cannot be completely discounted. Based on the assessment above, it is concluded that ϵ-caprolactone, oligomeric reaction products with 2,2’-oxydiethanol is unlikely to be a skin sensitiser; however this cannot be completely excluded due to the potential formation of dermal metabolites with sensitising potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 23 Jan 2018. Experimental completion date: 20 Feb 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Draft version July 2017 including LuSens test

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

A human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) up-stream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck). The test system employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity.
The assay included a cytotoxicity range finder test (CRFT) and three independent experiments (experiment I, II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the three experiments were deter-mined.
In the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.
Three experiments (experiment I, II and III) were performed in the same way. Experiment III serves only to confirm the results of experiment I and II.

Positive control results:

All control substances indicated the expected effect. No considerable reduction of the viability was de-tected (all values > 92 %). The positive control induced a clear effect with an induction value of 4.9 fold in comparison to the solvent control.

Key result

Parameter:

other: Fold induction

Value:

1.5

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:

Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SEN-SITISATION ARE-NRF2 LUCIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.

The following criteria for acceptability were met:

- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
- The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

The following test item concentrations showed a viability ≥ 70 %, were declared as valid and could therefore be evaluated for luciferase induction:

Experiment I: 269µg/mL, 323 µg/mL, 338 µg/mL, 465 µg/mL, 558 µg/mL.

Experiment II: 269µg/mL, 323 µg/mL, 338 µg/mL, 465 µg/mL.

Experiment III: 269µg/mL, 323 µg/mL, 338 µg/mL.

A substantial and reproducible dose-dependent and a statistically significant increase in luciferase induction was measured in the test item concentrations 323 µg/mL to 558 µg/mL in experiment I, 269 µg/mL to 465 µg/mL in experiment II and 269 to 388 µg/mL in experiment III. Here, the induction was equal or above threshold of 1.5 fold in comparison to the solvent control.

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol is considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.

Executive summary:

An in vitro study (conducted according to OECD Test Guideline 422D) has been carried out to evaluate the potential of the test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the CRFT.A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

A substantial and reproducible dose-dependent increase in luciferase induction above 1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both experiments.

Under the experimental conditions of this study, the test item,ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 30 May 2018. Experimental completion date: 12 June 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS:
-Source: Velaz, Prague, Czech Republic
- Females (nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 11 weeks
- Housing: Group housing (5 animals per cage)
- Diet: ad libitum
- Water: ad libitum
-Acclimation period. 5 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of analysis of diet as well as water is included in the raw data of the study.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 21-25
- Humidity (%): 50-60
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethyl sulphoxide

Concentration:

The doses tested were 0%, 25%, 50% and 100% of the substance.

No. of animals per dose:

5 animals per dose group

Details on study design:

PRE-SCREEN TEST
The pre-screen test was conducted under the same conditions as the main LLNA study but without the assessment of lymph node proliferation. A 100% concentration was used as undiluted test item. 25 µL of the test item was applied to the dorsum of each ear. The α−Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume.

The two female mice used in the pre-screen test were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%. For calculation of mean and S.D. values of body weights and ear thickness MS Excel was used.

MAIN TEST
5 female mice per treatment group were treated by daily topical application for 3 consecutive days with 25 µL of one of three concentrations of the test chemical (25%, 50% and 100% v/v) on the dorsum of each ear. The α−Hexylcinnamaldehyde (25%) as positive control and vehicle as a negative control were administrated in the same volume. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5 M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/animal and compared with DPM value from the vehicle control and expressed as the Stimulation Index (SI). The substance was regarded as a sensitizer in the LLNA if at least one concentration resulted in a SI value of 3 or greater and the data were not incompatible with a biological dose response.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The individual DPM data collected from component animals in the 25%, 50%, 100% as and negative control group were evaluated statistically.

The data were processed applying simple regression procedure as well as non-parametric multiple sample comparison (Kruskal-Wallis test) followed by pair–wise comparison (Mann-Whitney U-test). In the Simple Regression one-way ANOVA was carried out by breaking the total variance into variance due to model and residual.

Positive control results:

Treatment with α−Hexylcinnamaldehyde (25% in DMSO) in five animals served as positive control for the LLNA assay. The results generate calculated DPM and SI values of 1269.0 respective 9.09 (based on mean 10 lymph nodes). This is in compliance with the historical data of the test facility. The present study is therefore considered reliable.

Key result

Parameter:

SI

Value:

1.15

Test group / Remarks:

25% (v/v)

Key result

Parameter:

SI

Value:

1.44

Test group / Remarks:

50% (v/v)

Key result

Parameter:

SI

Value:

1.18

Test group / Remarks:

100% (v/v)

Cellular proliferation data / Observations:

EC3 CALCULATIONS:
EC3 value, which induce stimulation indices, is determined by linear interpolation of points on doseresponse curve, immediately above and below of SI value, according to the equation:
EC3=c+[(3-d)/(b-d)]x(a-c)
a – higher concentration, b – SI of higher concentration, c – lower concentration d – SI of lower concentration
If all points are below the stimulation index of three, no EC3 value can be stated. In the current study, the EC3 value could not be calculated since none of the tested concentrations induced a SI greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
No clinical observations were noted during the study.

BODY WEIGHTS:
The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. No marked changes of mean body weight were observed during the test.

Table 1: The mean lympho node weights, DPM and SI values

		Lymph node weight (g)	Number of lymph nodes	DPM	Mean SI	EC3 (%)
Control		0.064	10	139.6	-	-
Positive Control		0.0129	10	1269.0	9.09
ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol	25%	0.0064	10	161.0	1.15
	50%	0.0074	10	201.4	1.44
	100%	0.0064	10	164.8	1.18

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, ε-Caprolactone, oligomeric reaction products with 2,2'- oxydiethanol is not considered as a skin sensitizer under the condition of this LLNA study.

Executive summary:

The sensitization potential of “ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol” was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to

determine the allergic contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline 429, the test item was suspended in dimethyl sulfoxide (DMSO). The positive control (α-Hexylcinnamicaldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using the doses of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte

proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5 M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as

disintegrations per minute (DPM)/animal and compared with DPM value from the vehicle control and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25%, 50% and 100% v/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

In this study the mean SI values of 1.15, 1.44 and 1.18 were determined with the test item at concentrations of 25%, 50%, and 100% in DMSO, respectively. The EC3 value could not be calculated since none of the tested concentrations induced a mean SI value greater than the threshold value of 3.

The test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol is not considered as a skin sensitizer under the test conditions of this study.

Reference 5

Endpoint:

skin sensitisation: in chemico

Remarks:

OECD TG 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

(Q)SAR assessment

OECD QSAR Toolbox was used to identify structural alerts for ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol relating to skin sensitisation. Since the substance is oligomeric; modelling was therefore performed using a representative structure (DEG-2ECL). The representative structure of ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol does not show any structural alerts relating to skin sensitisation. OECD QSAR Toolbox was further used to predict the potential skin metabolites of the substance; the four skin metabolites were similarly screened for structural alerts relating to skin sensitisation. Three metabolites do not show any alerts; however one metabolite (Metabolite 1) shows a number of alerts due to the presence of an aldehyde (carbonyl) group. The extent of the dermal metabolism of ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol relating is unknown; however viable skin does possess esterase activity. The metabolite may therefore be produced in studies in vivo or in vitro using metabolically competent skin cells. The skin sensitisation potency of Metabolite 1 is unknown, but this is ranked as low potency (DPRA cysteine peptide depletion) or moderate potency (keratinocyte gene expression). The potential for a positive result in any assay will be determined both by the extent of metabolism and the potency of the metabolite. The potential for skin sensitisation does not appear to be very high, but cannot be completely discounted. Based on the assessment above, it is concluded that ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol is unlikely to be a skin sensitiser; however this cannot be completely excluded due to the potential formation of dermal metabolites with sensitising potential.

 

h-CLAT assay

This in vitro study was performed to assess the sensitising potential of the test item ε-caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, by quantifying changes in the expression levels of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. Three valid experiments with a treatment period of 24 hours were performed. In the experiments, the highest concentration (1000 µg/mL) was chosen based on cytotoxicity results obtained in the pre-test. A geometric series of seven dilutions was prepared. DMSO was used as solvent at a final concentration of 0.2% in the culture medium. 2,4-dinitrochlorobenzene (DNCB) was used as the positive control. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable.  No cytotoxic effect was detected in any experiment at any of the test concentrations. In Experiments I and II, the RFI of CD86 was <150%. In Experiment I the RFI of CD54 was <200%. In Experiment II, the RFI of CD54 was>200% at the highest test item concentration (1000 µg/mL). Therefore a third repetition was performed. In Experiment III the RFI of CD86 was <150% and the RFI of CD54 was <200%. As the majority of the results of the three individual runs is negative, the test item is considered as negative. All acceptance criteria were met and therefore, the study was considered valid. It can be concluded that, under the experimental conditions of this study, the test item, ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, have negative results in the h-CLAT and is therefore considered as not having the potential to activate dendritic cells and is a non-sensitiser.

 

LuSens assay

An in vitro study (conducted according to OECD Test Guideline 422D) has been carried out to evaluate the potential of the test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and three independent experiments (experiment I II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the three experiments were determined.

In the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the CRFT.A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control. A substantial and reproducible dose-dependent increase in luciferase induction above 1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in all experiments.

Under the experimental conditions of this study, the test item, ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

DPRA assay

This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. In this paticular case, this study was waived, as the substance is a UVCB substance. According to the test guideline on the In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), the test method should not be used for this type of substances.

In vivo skin sensitization study (LLNA assay)

The sensitization potential of “ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol” was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals. Based on the recommendations of the OECD Guideline 429, the test item was suspended in dimethyl sulfoxide (DMSO). The positive control (α-Hexylcinnamicaldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using the doses of 100 %. Based on the observations recorded in the Pre-screen tests, the concentration of 100 % was selected as top dose for the main test. In the main study, five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25%, 50% and 100%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive125I-iododeoxyuridine and 10^-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/animal and compared with DPM value from the vehicle control and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25%, 50% and 100% v/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

In this study the mean SI values of 1.15, 1.44 and 1.18 were determined with the test item at concentrations of 25%, 50%, and 100% in DMSO, respectively. The EC3 value could not be calculated since none of the tested concentrations induced a mean SI value greater than the threshold value of 3.

Based on the test result, the test item “ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol” is not considered as a skin sensitizer under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the overall conclusion from the available studies and supported by the QSAR evaluation, the test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol is not considered as a skin sensitizer.