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EC number: 949-790-7 | CAS number: -
Studies conducted to recognised testing guidelines with GLP certification
The result as generated by DEREK NEXUS is presented in Appendix 2. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 3.
Esacure 3644 is a UVCB of which the major component was assessed for skin sensitizing properties. DEREK NEXUS version 6.0.1 yielded four alerts for the major component of Esacure 3644 for skin sensitization based on the presence of an alpha,beta-unsaturated ketone, an alpha,beta-unsaturated ester, a dihydroxy coumarin, and a vinylic anisole. The dihydroxy coumarin and the vinylic anisole group may have the ability to act as a pre- or prohapten for skin sensitization.
For the alpha,beta-unsaturated ketone alert an EC3 of 13% was predicted based on data from 11 structurally related analogues (structural similarity of 16- 56%). For the alpha,beta-unsaturated ester alert an EC3 of 22% was predicted based on data from 10 structurally related analogues (structural similarity of 32- 58%). For the other two alerts no EC3 could be calculated due to insufficient data on analogues, and consequently the overall, worst case EC3 for Esacure 3644 cannot be determined.
Esacure 3644 was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table 1 and Figure 2-3. The results of the positive control are summarized in Table 2 and Figure 4-5. An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3. The individual raw data are presented in Appendix 3 and Appendix 4. The historical control data are presented in Appendix 5.
Two independent experiments were performed. The cells were in these experiments incubated with Esacure 3644 in a concentration range of 0.024 – 50 µg/ml (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• Esacure 3644 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 Esacure 3644. The Imax was 0.93 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.18 and the EC1.5 119 µM.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Esacure 3644. The Imax was 1.14 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.43 and the EC1.5 84 µM.
Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (119 µM and 84 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.18-fold and 2.43-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.0% and 8.1% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
The objective of this study was to evaluate the ability of Esacure 3644 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.
The study procedures described in this report were based on the most recent OECD guideline.
Batch M7-238-1807001 of Esacure 3644 was a light yellow solid. Esacure 3644 was dissolved in dimethyl sulfoxide at 5 mg/ml. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.024 – 50 µg/ml (2-fold dilution series). The highest test concentration was considered to be the limit of solubility. No precipitate was observed at any dose level tested. Two independent experiments were performed.
Both experiments passed the acceptance criteria:
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
Esacure 3644 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 in both experiments. The maximum luciferase activity induction (Imax) was 0.93-fold and 1.14-fold in experiment 1 and 2 respectively. Esacure 3644 is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/ml.
In conclusion, Esacure 3644 is classified as inconclusive under the experimental conditions described in this report.
Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 25 and 60% test item concentration, no signs of systemic toxicity were noted and no irritation was observed. Therefore, a 60% concentration was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 60% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
The auricular lymph nodes of the control animals and the animals treated at 60% were considered normal in size and the auricular lymph nodes of the animals treated at 10% and 25% were considered to be slightly enlarged compared to normal. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 60% were 887, 703 and 731 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test item concentrations 10, 25 and 60% were 1.7, 1.4 and 1.4, respectively.
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 60%, Esacure 3644 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 60%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, Esacure 3644 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment (study no 20186176) and a KeratinoSensTM assay (study no 20186178).
Results of studies performed
DEREK NEXUS version 6.0.1 yielded four alerts for the major component of Esacure 3644 for skin sensitization based on the presence of an alpha,beta-unsaturated ketone, an alpha,beta-unsaturated ester, a dihydroxy coumarin, and a vinylic anisole. The dihydroxy coumarin and the vinylic anisole group may have the ability to act as a pre- or prohapten for skin sensitization.For two of the four alerts no EC3 could be calculated due to insufficient data on analogues, and consequently the overall, worst case EC3 for Esacure 3644 cannot be determined. In conclusion, the main component of Esacure 3644 is predicted to be sensitizing to the skin (plausible). The overall, worst case EC3 value could not be determined.
A valid KeratinoSensTM assay was performed according to OECD 442D and GLP. For the KeratinoSensTM assay Esacure 3644 was dissolved in dimethyl sulfoxide to a final concentration of 5 mg/mL (yellow solution). Test concentrations of 0.024 – 50 μg/mL were used in the experiments. The highest test concentration was considered to be the limit of solubility. Two independent experiments were performed. No precipitation and cytotoxicity was observed at all dose levels tested. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 0.93-fold and 1.14-fold in experiment 1 and 2 respectively. Esacure 3644 is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/ml.
The DEREK assessment predicted Esacure 3644 to be a skin sensitizer based on four different groups present. However, no overall worst case EC3 could be determined. The KeratinoSensTM assay showed no activation of keratinocytes up to and including the highest concentration tested (limit of solubility). As the top concentration was too low to determine a reliable result in the KeratinoSensTM assay the outcome was regarded as inconclusive.
As DEREK predicted that metabolism might play a role in the sensitizing ability of Esacure 3644, and the KeratinoSensTM assay has only limited metabolic capacity, the absence of any induction might also be false. Based on these results additional testing is needed however performing a U-SENSTM assay as a next step would not lead to a definitive conclusion on the skin sensitizing properties of Esacure 3644. In case of a positive outcome the substance is regarded as a sensitizer and potency would need to be determined in vivo. As metabolism may play a role in skin sensitization of Esacure 3644, a negative outcome of the U-SENSTM assay would still require an in vivo assay to be performed, since the U-SENSTM assay only has limited metabolic capacity and may be false negative.
Since the current data set is not adequate for classification and risk assessment on the endpoint skin sensitization and further in vitro testing would not lead to a conclusion, it is considered justified to perform an in vivo study to assess the skin sensitizing properties of Esacure 3644.
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