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Description of key information

A DEREK assessment, DPRA assay and KeratinoSensTMassay were performed in accordance withSection 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 andthe strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
In silico assessment
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
2018
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 adequate and reliable documentation / justification
Justification for type of information:
An in silico assesment is mentioned in the ECHA guidance as one of the non-animal data that may be provided as weight of evidence.
Principles of method if other than guideline:
An in silico assessment was performed with DEREK NEXUS version 6.0.1.
GLP compliance:
no
Justification for non-LLNA method:
Since 11 October 2016 it is legally required to consider all available information for the endpoint skin sensitisation and to use a non in vivo test strategy based on in chemico, in silico and in vitro skin tests combined with a WoE. An in vivo test (LLNA) is only allowed as last resort. DEREK results are adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.
Key result
Run / experiment:
other: Delta Octalactone
Parameter:
other: Prediction on skin sensitization
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Delta Octalactone does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.
Interpretation of results:
study cannot be used for classification
Remarks:
(study is part of a weight of evidence approach and is not used for classification on its own)
Conclusions:
Delta Octalactone is predicted to be not sensitizing to the skin.
Executive summary:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, Delta Octalactone does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 April 2018 - 01 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of a weight of evidence approach.
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of a weight of evidence approach.
Details on the study design:
TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 23.54 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1655 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

DATA EVALUATION:
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 1), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.
Key result
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: Mean SPCC depletion(%)
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 0.8%
Positive controls validity:
valid
Remarks:
Mean percentage SPCC: 74.0% ± 0.2%.
Remarks on result:
other: SD: 0.3%
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: Mean percentage SPCC:
Value:
2.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
CV between reference controls: 1.3%
Positive controls validity:
valid
Remarks:
Mean percentage SPCL: 53.2% ± 1.8%.
Remarks on result:
other: SD: 0.7%
Other effects / acceptance of results:
All acceptability criteria were met and therefore the DPRA was considered to be valid.
See Table 2 & 3 "any other information on results incl. tables" for acceptibility criteria.

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay 

Lysine reactivity assay

Acceptability criteria

Results for

SPCC

Acceptability criteria

Results for

SPCL

Correlation coefficient (r2) standard calibration curve 

>0.99

0.996

>0.99

0.997

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05 

0.508 ± 0.006

0.50 ± 0.05

0.498 ± 0.007

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.505 ± 0.005

0.50 ± 0.05

0.498 ± 0.004

CV (%) for RC samples B and C

<15.0

0.8

<15.0

1.3

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

74.0

40.2-69.0

53.2

SD of peptide depletion cinnamic aldehyde (%)

<14.9

0.2

<11.6

1.8

SD of peptide depletion for the test item (%)

<14.9

0.3

<11.6

0.7

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item 

SPCC depletion 

SPCL depletion

Mean of

SPCC and

SPCL

depletion

DPRA prediction and reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

Tetrahydro6propyl-2Hpyran-2-one

(delta octalactone)

1.2%

±0.3%

2.5%

±0.7%

1.9%

Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:
other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation
Conclusions:
Delta octalactone was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2018 - 29 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The purpose of this study was to evaluate the ability of Polyambrol to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.
Specific details on test material used for the study:
Specific gravity: 0.998
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

CONTROLS:
Positive control: ethylene dimethacrylate glycol (purity: 98.3%), 7.8-250 µM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25). One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+6 in experiment 1 and P+8 in experiment 2.

TEST ITEM PREPARATION:
The test item was dissolved/suspended in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test item concentrations were used within 3.5 hours after preparation.

TEST CONCENTRATIONS:
- Both experiments: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM

MEDIA:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

TREATMENT OF CELLS:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours in a humid atmosphere of 80 - 100% (actual range 72 – 95 %) at 37.0 ± 1.0°C (actual range 35.6 – 37.2°C), in the presence of 5% ± 0.5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Positive control results:
The EC1.5 of the positive control was between 26 and 125 μM (59 μM and 46 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (3.98-fold and 4.59-fold in experiment 1 and 2, respectively).
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.12
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
- In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.
- In both experiments, the test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item in both experiments. The Imax were 1.17 and 1.12 in experiment 1 and experiment 2, respectively. From these Imax values, no EC1.5 could be calculated.


Both experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (26 μM and 46 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (3.98-fold and 4.59-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (12% and 10% in experiment 1 and 2, respectively).

Table 1 Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp 1 luminescence

1.13

1.00

1.06

1.14

1.17

1.14

1.12

1.15

1.09

0.95

0.90

0.80

Exp 1 viability (%)

98.9

106.6

99.5

96.6

98.2

91.1

93.2

89.3

95.2

101.8

103.7

95.7

Exp 2 luminescence

1.09

0.83

1.00

1.08

1.10

1.12

1.12

1.11

1.02

1.03

1.06

0.88

Exp 2 viability (%)

115.4

122.4

99.8

102.3

100.6

97.8

95.8

95.1

96.7

98.3

97.9

102.5

Table 2 Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.22

1.28

1.60***

1.86***

2.67***

3.98***

Exp 1 viability (%)

98.7

94.7

107.5

110.8

110.9

106.5

Exp 2 luminescence

1.03

1.13

1.31

1.72***

2.32***

4.59***

Exp 2 viability (%)

105.6

109.2

106.2

107.2

107.1

69.7

***p<0.001 Student’s t test

Table 3 Overview EC1.5, Imax, IC30 and IC50 Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.17

NA

NA

Test item Experiment 2

NA

1.12

NA

NA

Pos Control Experiment 1

26

3.98

NA

NA

Pos Control Experiment 2

46

4.59

NA

NA

NA = Not applicable

Interpretation of results:
study cannot be used for classification
Remarks:
This study is part of a weight of evidence approach on which the classification is based.
Conclusions:
The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations two independent experiments. The maximum luciferase activity induction (Imax) was 1.17-fold and 1.12-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens(TM) assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.
Based on the outcome of this KeratinoSens™ assay performed according to OECD guideline and GLP principles, Tetrahydro-6propyl-2H-pyran-2-one (delta octalactone) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For skin sensitization, substance analogue Delta octalactone was tested. The conclusion on this endpoint was drawn following a weight-of-evidence approach:

1.    DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for Delta octalactone. It is of note that the substance does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

2.    Delta octalactone was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.  

3.    Delta octalactone was classified as negative in a KeratinoSens™ assay (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Based on this data-set it is concluded that there are no indications that Delta octalactone has skin sensitizing properties.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data are considered sufficient to conclude that Delta octalactone does not have to be classified for skin sensitizing properties according to Regulation 1272/2008 and amendments.