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Administrative data

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

Referenceopen allclose all

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.

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.

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).

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.

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.