Registration Dossier

Administrative data

Description of key information

In Silico: The test item was predicted to be non-sensitising to skin (DEREK NEXUS version 6.0.1).

In chemico: The test item 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 (OECD 442C).

In vitro: The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions of the KeratinoSens assay (OECD 442D).

Weight of evidence: Based on a weight of evidence from the studies performed and the information publicly available on lactic acid, the test item is considered to be not sensitising to skin (expert opinion).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
16 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
See Appendix 6 (attached).
Qualifier:
no guideline required
Principles of method if other than guideline:
Prediction on the potential for skin sensitization of the test item with the in silico model DEREK NEXUS.
GLP compliance:
no
Details on study design:
INTRODUCTION
- The objective of this study was to obtain a prediction on the potential for skin sensitisation of the test item with the in silico model DEREK NEXUS.
- In this assessment version 6.0.1 of DEREK NEXUS was used.

BACKGROUND / SCOPE
- DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation
based on the presence of molecular substructures. LHASA (see Appendix I, attached) has inserted validation comments for the skin sensitisation alerts.
- The level of likelihood of a structure being sensitising to skin is expressed in terms of:
(i) Certain: There is proof that the proposition is true.
(ii) Probable: There is at least one strong argument that the proposition is true and there are no arguments against it.
(iii) Plausible: The weight of evidence supports the proposition.
(iv) Equivocal: There is an equal weight of evidence for and against the proposition.
- The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment. DEREK NEXUS contains expert-derived functionality to provide negative predictions for skin sensitisation. This functionality further evaluates compounds which do not fire an alert for skin sensitisation. The query compound is compared to the Lhasa skin sensitisation negative predictions dataset, producing the following outcomes:
(a) Where all features in the molecule are found in accurately classified compounds from
the dataset, a prediction of Non-Sensitiser is displayed.
(b) For those compounds where features in the query are found in non-alerting sensitisers in the Lhasa dataset, the prediction remains Non-Sensitiser, but these Misclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
(c) In cases where features in the query are not found in the Lhasa dataset, the prediction remains Non-Sensitiser, but these Unclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
- If a substance is predicted to be a skin sensitiser, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present). The EC3 is the estimated concentration needed to produce a stimulation index of 3 and is calculated using the equation EC3Q = MWQ /(Σ ωNN / Σ TNN) where MW = molecular weight; T = Tanimoto similarity score; ω = weighting factor = (MWNN/EC3) * TNN; Q = query compound; NN = nearest neighbour.

SUBSTANCE ASSESSESSED
- The substance contained four main components, which represented 99 % of the test item evaluated (see attachment).
Key result
Parameter:
other: prediction of skin sensitisation
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
RESULTS
- The result as generated by DEREK NEXUS is presented in Appendices 2 to 5 (attached).
- The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are presented in Appendix 6 (attached).
- DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for each of the four components and the substance is predicted to be not sensitising to the skin.
- The query structures do not match any structural alerts or examples for skin sensitisation in DEREK.
- Additionally, the query structures do not contain any unclassified or misclassified features and the test item is consequently predicted to be a non-sensitiser.
Interpretation of results:
GHS criteria not met
Conclusions:
DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for each of the four components and the test item was predicted to be not sensitising to the skin. The query structures do not match any structural alerts or examples for skin sensitisation in DEREK. Additionally, the query structures do not contain any unclassified or misclassified features and the test item is consequently predicted to be a non-sensitiser.
Executive summary:

METHOD

The objective of the study was to obtain a prediction on the potential for skin sensitisation of the test item with the in silico model DEREK NEXUS version 6.0.1. DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. The level of likelihood of a structure being sensitising to skin is expressed in terms of: Certain (there is proof that the proposition is true); Probable (there is at least one strong argument that the proposition is true and there are no arguments against it); Plausible (the weight of evidence supports the proposition); Equivocal (there is an equal weight of evidence for and against the proposition). The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment. DEREK NEXUS contains expert derived functionality to provide negative predictions for skin sensitisation. This functionality further evaluates compounds which do not fire an alert for skin sensitisation. The query compound is compared to the Lhasa skin sensitisation negative predictions dataset, producing the following outcomes (i) Where all features in the molecule are found in accurately classified compounds from the dataset, a prediction of Non-Sensitiser is displayed (ii) For those compounds where features in the query are found in non-alerting sensitisers in the Lhasa dataset, the prediction remains Non Sensitiser, but these Misclassified features are highlighted to enable the negative prediction to be verified by expert assessment (iii) In cases where features in the query are not found in the Lhasa dataset, the prediction remains Non-Sensitiser, but these Unclassified features are highlighted to enable the negative prediction to be verified by expert assessment. If a substance is predicted to be a skin sensitiser, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental datafrom the closest structurally-related substances (at least 3 substances should be present).

 

RESULTS

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for each of the four components and the test item was predicted to be not sensitising to the skin. The query structures do not match any structural alerts or examples for skin sensitisation in DEREK. Additionally, the query structures do not contain any unclassified or misclassified features and the test item is consequently predicted to be a non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2018 to 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
temporary deviations to temperature and humidity with no impact on results or integrity of the study
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on study design:
VEHICLE
- Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- No correction was made for the composition/purity of the test item.
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 40 mg/mL (clear colourless solution). The 100-fold dilution of the 40 mg/mL DMSO stock in DMEM glutamax formed also a clear solution (400 μg/mL). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL (clear colourless 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 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.
- 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 hours after preparation. Any residual volumes were discarded.

PREPARATION OF THE POSITIVE CONTROL
- The positive control used in the case of KeratinoSens is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

PREPARATION OF THE VEHICLE CONTROL
- The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per
plate.

BLANK
- On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM
- A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens cell line). The KeratinoSens cell line was generated by and obtained from Givaudan (Duebendorf, Switserland).
- 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 from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
- Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
- Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
- Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 71 - 98 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data (attached) these deviations are considered not to affect the study integrity.

SUBCULTURING
- Cells were subcultured upon reaching 80 to 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).

PLATING OF CELLS
- For testing, cells were 80 to 90% confluent.
- 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+10 in experiment 1, P+12 in experiment 2 and P+15 in experiment 3.

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 items were added.
- Three wells per plate were left empty (no cells and no treatment) to assess background values.
- The treated plates were covered with foil and then incubated for about 48 hours at 37 ± 1.0 °C in the presence of 5% CO2.
- A total of 3 valid experiments were performed.

LUCIFERASE ACTIVITY MEASUREMENT
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together.
- The assay plates were removed from the incubator and the medium 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 TECAN Infinite M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT
- For the KeratinoSens 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; Sigma, Zwijndrecht, The Netherlands) 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 (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
- The KeratinoSens test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
- All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- The following parameters are calculated in the KeratinoSens test method:
i) The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
ii) The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
iii) The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
- Fold luciferase activity induction is calculated using the equation Fold Induction = (Lsample – Lblank) / (Lsolvent – Lblank) where Lsample = luminescence reading in the test chemical well; Lblank = luminescence reading in the blank well containing no cells and no treatment; Lsolvent = average luminescence reading in the wells containing cells and vehicle (negative) control. The overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
- The EC1.5 is calculated by linear interpolation using the equation EC1.5 = (Cb – Ca) * [(1.5 - Ia) / (Ib – Ia)] + Ca where Ca = the lowest concentration in μM with > 1.5 fold induction; Cb = the highest concentration in μM with < 1.5 fold induction; Ia = the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells); Ib = the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells).
- Viability is calculated using the equation Viability = (Vsample – Vblank) / (Vsolvent – Vblank) * 100 where Vsample = the MTT-absorbance reading in the test chemical well; Vblank = the MTT-absorbance reading in the blank well containing no cells and no treatment; Vsolvent = the average MTT-absorbance reading in the wells containing cells and vehicle
(negative) control.
- Control IC50 and IC30 are calculated by linear interpolation using the equation ICx = (Cb – Ca) * [((100-x) – Va) / (Vb – Va)] + Ca where x = the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30); Ca = the lowest concentration in μM (or μg/mL) with > x% reduction in viability; Cb = the highest concentration in μM (or μg/mL) with < x% reduction in viability; Va = the % viability at the lowest concentration with > x% reduction in viability; Vb = the % viability at the highest concentration with < x% reduction in viability. The overall IC50 and IC30 are calculated as the mean of the individual repetitions.
- In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the vehicle (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

DATA INTERPRETATION
- A KeratinoSens prediction is considered positive if the following 4 conditions are all met in
2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered
negative (see Figure 1, attached):
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test).
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration).
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW).
4. There is an apparent overall dose-response for luciferase induction.
- Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.

CRITICAL COMPUTERISED SYSTEMS
- REES Centron version SQL 2.0: Temperature and humidity (laboratory facilities) data collection.
- Magellan Tracker version 7.0: Optical Density and Luminescence Measurement.
Positive control results:
- See Table 2 and Figures 5 to 7 (attached)
Key result
Parameter:
other: fold induction
Run / experiment:
1 to 3
Value:
> 1.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: percentage cell viability relative to vehicle control
Run / experiment:
1 to 3
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS
- The test item 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 summarised in Table 1 and Figures 2 to 4 (attached).
- The results of the positive control are summarised in Table 2 and Figures 5 to 7 (attached).
- An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3 (attached).
- Three independent experiments were performed. The cells were in these experiments
incubated with test item in a concentration range of 0.20 to 400 μg/mL (2-fold dilution steps) for 48 hours. 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.

EXPERIMENT 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 7.0 μg/mL and the calculated IC50 was 8.6 μg/mL.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with test item. The Imax was 1.47 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 2.83 and the EC1.5 48 μM.

EXPERIMENT 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 14 μg/mL and the calculated IC50 was 17 μg/mL.
- A dose related luminescence activity induction was observed after treatment with test item. The Imax was 1.83 and the EC1.5 9.1 μg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.62 and the EC1.5 85 μM.
- Since the results of the first two experiments were not concordant (no biologically relevant induction in the first experiment and positive result in the second experiment) a third experiment was performed.

EXPERIMENT 3
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 13 μg/mL and the calculated IC50 was 16 μg/mL. At 6.3 μg/mL a decrease in the viability was observed (viability of 61.1%), however, since at 13 μg/mL the viability was higher than 70% it was considered that this decrease was caused by variation in the data and the decrease was considered not relevant for the cytotoxicity endpoint.
- A dose related luminescence activity induction was observed after treatment with test item. The Imax was 1.62 and the EC1.5 10 μg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.92 and the EC1.5 36 μM.

ACCEPTANCE CRITERIA
- All 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 (48 μM, 85 μM and 36 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold, 2.62-fold and 2.92-fold in experiment 1, 2 and 3 respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4%, 7.8% and 6.4% in experiment 1, 2, and 3 respectively).
- Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
other: positive
Conclusions:
The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

GUIDELINE

The study was conducted in accordance with OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).

 

METHODS

The objective of this study was to evaluate the ability of test item to activate the

antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The clear, colourless, test material was dissolved in dimethyl sulfoxide at 40 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 0.20 to 400 μg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed since the results of the test item in the first two experiments were not concordant.

 

RESULTS

All 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 (48 μM, 85 μM and 36 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold, 2.62-fold and 2.92-fold in experiment 1, 2 and 3 respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4%, 7.8% and 6.4% in experiment 1, 2, and 3 respectively). Overall it was concluded that the test conditions were adequate and that the test system functioned properly.

 

The test item showed toxicity (IC30 values of 7.0 μg/mL, 14 μg/mL and 13 μg/mL and IC50 values of 8.6 μg/mL, 17 μg/mL and 16 μg/mL in experiment 1, 2 and 3, respectively). In the first experiment no biologically relevant induction of the luciferase activity was observed. In the second and third experiment, a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.1 μg/mL and 10 μg/mL in experiment 2 and 3, respectively) was measured. The maximum luciferase activity induction (Imax) was 1.47-fold, 1.83-fold and 1.62-fold in experiment 1, 2 and 3 respectively. The test material was classified as positive in the KeratinoSens assay since positive results (>1.5- fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control in 2 out of 3 experiments.

 

CONCLUSION

The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in chemico
Remarks:
Direct Peptide Reactivity Assay (DPRA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2018 to 07 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Key result
Parameter:
other: mean of SPCC and SPLC depletion
Remarks:
percent
Value:
1.8
Remarks on result:
other: negative (no or minimal reactivity)

SOLUBILITY ASSESSMENT OF THE TEST ITEM

- At a concentration of 100 mM, the test item was not soluble in MQ and ACN:MQ (1:1, v/v) but was soluble in ACN and isopropanol. As ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this DPRA study.

 

CYSTEINE REACTIVITY ASSAY

- The reactivity of test item towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24 hours of incubation at 25 ± 2.5 °C.

- Representative chromatograms of CCcys-209188/A and 209188/A-cys samples are presented in Appendix 4 (attached). An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (see Appendix 3, attached).

 

ACCEPTABILITY OF THE CYSTEINE REACTIVITY ASSAY

- The SPCC standard calibration curve is presented in Figure 1 (see Appendix 2, attached). The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9991. Since the r2 was > 0.99, the SPCC standard calibration curve was accepted.

- The results of the Reference Control samples A and C are presented in Table 4 (see Appendix 3, attached). The mean peptide concentration of Reference Controls A was 0.540 ± 0.026 mM while the mean peptide concentration of Reference Controls C was 0.515 ± 0.051 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

- The SPCC peak areas for Reference controls B and C are presented in Table 5 (see Appendix 3, attached). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 6.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

- The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (see Appendix 3, attached). The mean area ratio (A220/A258) of the Reference Control samples was 19.26. The mean A220/A258 ratio ± 10 % range was 17.33-21.19. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

- The results of the positive control cinnamic aldehyde are presented in Table 7 (see Appendix 3, attached). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference

Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 78.6% ± 1.0 %. This was within the acceptance range of 60.8 % to 100 % with a SD that was below the maximum (SD < 14.9 %).

 

RESULTS OF THE CYSTEINE REACTIVITY ASSAY FOR THE TEST ITEM

- Preparation of a 100 mM test item stock solution in ACN showed that the test item was dissolved completely. After incubation a phase separation, visible as an oily layer on top of the solution in the vial, was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

- The results of the cysteine reactivity assay for the test item are presented in Table 8 (see Appendix 3, attached). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4, attached). This demonstrated that there was no co-elution of the test item with SPCC. For the 209188/A-cys samples, the mean SPCC A220/A258 area ratio was 19.03. Since this was within the 17.33-21.19 range, this again indicated that there was no coelution of the test item with SPCC.

- The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 3.6 % ± 6.2 %.

 

LYSINE REACTIVITY ASSAY

- The reactivity of test item towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 23.5 hours of incubation at 25 ± 2.5 °C. Representative chromatograms of CClys-209188/A and 209188/A-lys samples are presented in Appendix 4 (attached). An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (see Appendix 3, attached).

 

ACCEPTABILITY OF THE LYSINE REACTIVITY ASSAY

- The SPCL standard calibration curve is presented in Figure 2 (see Appendix 2, attached). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.996. Since the r2 was > 0.99, the SPCL standard calibration curve was accepted.

- The results of the Reference Control samples A and C are presented in Table 10 (see Appendix 3, attached). The mean peptide concentration of Reference Controls A was 0.493 ± 0.009 mM, while the mean peptide concentration of Reference Controls C was 0.482 ± 0.019 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

- The SPCL peak areas for Reference controls B and C are presented in Table 11 (see Appendix 3, attached). The CV of the peptide areas for the nine Reference Controls B and C was 3.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

- The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (see Appendix 3, attached). The mean area ratio (A220/A258) of the Reference Control samples was 15.40. The mean A220/A258 ratio ± 10% range was 13.86-16.94. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

- The results of the positive control cinnamic aldehyde are presented in Table 13 (see Appendix 3, attached). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.2 % ± 2.4 %. This was within the acceptance range of 40.2 % to 69.0 % with a SD that was below the maximum (SD < 11.6 %).

 

RESULTS OF THE LYSINE REACTIVITY ASSAY FOR THE TEST ITEM

- Preparation of a 100 mM test item stock solution in ACN showed that the test item was dissolved completely. After incubation a phase separation, visible as an oily layer on top of the solution in the vial, was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the

peptide.

- The results of the lysine reactivity assay for the test item are presented in Table 14

(see Appendix 3, attached). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4, attached). This demonstrated that there was no co elution of the test item with SPCL.

- For the 209188/A-cys samples, the mean SPCL A220/A258 area ratio was 15.38. Since this was within the 13.86-16.94 range, this again indicated that there was no co-elution of the test item with SPCL.

- The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 0.0% ± 0.0%.

 

DPRA PREDICTION AND REACTIVITY CLASSIFICATION

- Upon preparation of the SPCC and SPCL test item samples, no precipitate was observed in any of the samples, however, after incubation of the SPCC and SPCL test item samples, a phase separation, visible as an oily layer on top of the solution in the vial, was observed in the co-elution control (CC) and test item samples. In this case it is not possible to be sure how much test item remained in the solution to react with the peptide.

- An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 3.6 % SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.8 % and as a result the test item 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. Therefore, the test item was considered to be negative in the DPRA.

SPCC AND SPCL DEPLETION – DPRA PREDICTION AND REACTIVITY CLASSIFICATION FOR THE TEST ITEM

Material

SPCC depletion mean

SPCC depletion±SD

SPCL depletion mean

SPCL depletion ± SD

DPRA prediction and reactivity classification*

Test Item

3.6 %

± 6.2 %

0.0 %

± 0.0 %

Negative: No or minimal reactivity

SD = Standard Deviation

* = Cysteine 1:10 / Lysine 1:50 prediction model

Interpretation of results:
GHS criteria not met
Conclusions:
Since all acceptability criteria were met this DPRA is considered to be valid. The test item 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. However, since phase separation was observed after the incubation period for both SPCC and SPCL, it is not possible to be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
Executive summary:

GUIDELINE

The objective of the study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). The design of the study was based on OECD Guideline for the Testing of Chemicals, Guideline 442C In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) (4 February 2015).

 

METHODS

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 °C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

 

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitisers.

 

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

RESULTS

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

 

Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples, however, after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

 

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were reported. In the cysteine reactivity assay the test item showed 3.6% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.8% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

CONCLUSION

Since all acceptability criteria were met this DPRA is considered to be valid. The test item 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. However, since phase separation was observed after the incubation period for both SPCC and SPCL, it is not possible to be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Endpoint:
skin sensitisation: in vitro
Remarks:
weight of evidence
Type of information:
other: expert assessment of in silico, in chemico and in vitro results
Adequacy of study:
key study
Study period:
April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline required
Principles of method if other than guideline:
Expert consideration of available data describing skin sensitisation potential.
GLP compliance:
no
Type of study:
other: expert opinion
Key result
Parameter:
other: in silico prediction
Remarks:
DEREK NEXUS version 6.0.1
Remarks on result:
other: negative
Key result
Parameter:
other: KeratinoSens assay
Remarks on result:
other: negative
Key result
Parameter:
other: Direct Peptide Reactivity Assay (DPRA)
Remarks on result:
other: negative
Other effects / acceptance of results:
RESULTS OF STUDIES PERFORMED
- No data were available that would preclude performance of the studies to determine the potential for skin sensitisation. Therefore, STEP 1 studies were performed, i.e. DEREK assessment (project no 20140636); DPRA assay (project no 20140637) and KeratinoSens assay (project no 20140638).
- DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for each of the four components. The test item is predicted to be not sensitising to the skin. The query structures do not match any structural alerts or examples for skin sensitisation in DEREK. Additionally, the query structures do not contain any unclassified or misclassified features and the test item is consequently predicted to be a non-sensitiser.
- A valid DPRA test was performed according to OECD 442C and GLP. For the DPRA assay The test item was dissolved in acetonitrile at 100 mM. No co-elution of the test item with SPCC or SPCL was observed. In the cysteine reactivity assay the test item showed 3.6% SPCC depletion, and in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.8%. As a result, the test item was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model and was considered to be negative in the DPRA. However, phase separation was observed after the incubation period (not before) for both SPCC and SPCL incubations, and the negative result should be interpreted with due caution.
- A valid KeratinoSens assay was performed according to OECD 442D and GLP. For the KeratinoSensTM assay, test item was dissolved in DMSO to a final concentration of 40 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 0.20 – 400 µg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed since the results of the test item in the first two experiments were not concordant. The test item showed toxicity with an IC30 of 7.0 μM and an IC50 of 8.6 μM in experiment 1, an IC30 of 14 μM and IC50 value of 17 μM in experiment 2, and an IC30 of 13 μM and IC50 value of 16 μM in experiment 3. In the first experiment no biologically relevant induction of the luciferase activity was observed. In the second and third experiment, a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.1 µg/mL and 10 µg/mL in experiment 2 and 3, respectively) was measured. The maximum luciferase activity induction (Imax) was 1.47-fold, 1.83-fold and 1.62-fold in experiment 1, 2 and 3 respectively. In conclusion, the test item is classified as positive in the KeratinoSens assay, since positive results (> 1.5-fold induction) were observed at test concentrations <200 µg/mL with a cell viability of > 70 % compared to the vehicle control in 2 out of 3 experiments.
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In silico

The objective of the study was to obtain a prediction on the potential for skin sensitisation ofthe test item with the in silico model DEREK NEXUS version 6.0.1. DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecularsubstructures. The level of likelihood of a structure being sensitising to skin is expressed in terms of:Certain (there is proof that the proposition is true); Probable (there is at least one strong argumentthat the proposition is true and there are no arguments against it); Plausible (the weight of evidence supports the proposition); Equivocal (there is an equal weight of evidence for and against theproposition). The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which areevaluated as being equivocal or greater was used in this assessment. DEREK NEXUS contains expertderived functionality to provide negative predictions for skin sensitisation. This functionality furtherevaluates compounds which do not fire an alert for skin sensitisation. The query compound iscompared to the Lhasa skin sensitisation negative predictions dataset, producing the followingoutcomes (i) Where all features in the molecule are found in accurately classified compounds fromthe dataset, a prediction of Non-Sensitiser is displayed (ii) For those compounds where features in the query are found in non-alerting sensitisers in the Lhasa dataset, the prediction remains NonSensitiser, but these Misclassified features are highlighted to enable the negative prediction to beverified by expert assessment (iii) In cases where features in the query are not found in the Lhasadataset, the prediction remains Non-Sensitiser, but these Unclassified features are highlighted toenable the negative prediction to be verified by expert assessment. If a substance is predicted to bea skin sensitiser, its potency is predicted by DEREK NEXUS by calculating an EC3 value based onexperimental datafrom the closest structurally-related substances (at least 3 substances should be present).

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for each of thefourcomponents and the test item was predicted to be not sensitising to the skin. The query structures do not match any structural alerts or examples for skin sensitisation in DEREK. Additionally, the query structures do not contain any unclassified or misclassified featuresand the test item is consequently predicted to be a non-sensitiser.

In chemico

Direct Peptide Reactivity Assay (DPRA)

GUIDELINE

The objective of the study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). The design of the study was based on OECD Guideline for the Testing of Chemicals, Guideline 442C In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) (4 February 2015).

 

METHODS

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 °C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

 

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitisers.

 

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.

 

RESULTS

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

 

Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples, however, after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

 

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion were reported. In the cysteine reactivity assay the test item showed 3.6% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.8% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

 

CONCLUSION

Since all acceptability criteria were met this DPRA is considered to be valid. The test item 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. However, since phase separation was observed after the incubation period for both SPCC and SPCL, it is not possible to be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

In vitro

KeratinoSens Assay

GUIDELINE

The study was conducted in accordance with OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).

 

METHODS

The objective of this study was to evaluate the ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The clear, colourless, test material was dissolved in dimethyl sulfoxide at 40 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 0.20 to 400 μg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed since the results of the test item in the first two experiments were not concordant.

 

RESULTS

All 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 (48 μM, 85 μM and 36 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold, 2.62-fold and 2.92-fold in experiment 1, 2 and 3 respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4%, 7.8% and 6.4% in experiment 1, 2, and 3 respectively). Overall it was concluded that the test conditions were adequate and that the test system functioned properly.

 

The test item showed toxicity (IC30 values of 7.0 μg/mL, 14 μg/mL and 13 μg/mL and IC50 values of 8.6 μg/mL, 17 μg/mL and 16 μg/mL in experiment 1, 2 and 3, respectively). In the first experiment no biologically relevant induction of the luciferase activity was observed. In the second and third experiment, a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.1 μg/mL and 10 μg/mL in experiment 2 and 3, respectively) was measured. The maximum luciferase activity induction (Imax) was 1.47-fold, 1.83-fold and 1.62-fold in experiment 1, 2 and 3 respectively. The test material was classified as positive in the KeratinoSens assay since positive results (>1.5- fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control in 2 out of 3 experiments.

 

CONCLUSION

The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Weight of evidence

The objective was to evaluate whether sufficient information is available to meet the information requirements for skin sensitisation of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification in accordance with Regulation (EC) No 1272/2008 (CLP) and related amendments. A weight of evidence approach, according to Annex XI, sections 1.2-1.5, to the REACH Regulation, was used. The design of the study was based on ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a Endpoint specific guidance v.6.0 July 2017, paragraph 7.3.

 

DATA ASSESSED

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

 

RESULTS

The DEREK and DPRA assay gave a negative result and the marginally positive result of the KeratinoSens assay is considered doubtful in the light of the negative results of the other studies. From data provided by the sponsor it could be concluded that the starting materials lactic acid and lauryl alcohol are not skin sensitisers. Reaction of a carboxylic acid with an alcohol to yield an ester is not going to change the skin sensitising properties.

 

CONCLUSION

Based on a weight of evidence from the studies performed and the information publicly available on lactic acid, the test item is considered to be not sensitising to skin and does not need to be classified according to Regulation 1272/2008 and subsequent amendments.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on a weight of evidence from the studies performed and the information publicly available on lactic acid, the test item is considered to be not sensitising to skin, and does not need to be classified according to Regulation 1272/2008 and subsequent amendments.