5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

Endpoint summary

Description of key information

In silico - DEREK calculation

The substance is predicted to be not sensitising to the skin, but this prediction should be considered with care.

In Chemico, Direct Peptide Reactivity Assay (Reinen, 2018)

Since no suitable solvent compatible with the DPRA to dissolve the test material at the desired concentration could be found, no main experiment could be performed. Therefore, the reactivity of the test material towards SPCC and SPCL could not be determined.

In vitro, Keratinosens Assay (Woutersen, 2018)

Under the conditions of this study, the test material 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.

Weight of Evidence (Pelgrom, 2018)

Based on the in vitro tests performed no definite conclusion on skin sensitising potential and/or potency can be drawn for the substance. In the absence of a reliable and acceptable in vitro skin sensitising test with inclusion of potency testing, an in vivo test (LLNA) needs to be performed.

In vivo Local Lymph Node Assay (van Sas, 2018)

Based on the results of the LLNA study it should be considered to regard the test material as a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 February 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:

1. SOFTWARE
DEREK NEXUS - skin sensitisation

2. MODEL (incl. version number)
DEREK NEXUS version 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
C1=C(C(=CC(=C1C2=CC(=(C=C2C)OI)C(C)C)C)OI)C(C)C

- Molecular Mass: 549.9866

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 77 %.

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitisation alerts.
The level of likelihood of a structure being sensitizing 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.
If a substance is predicted to be no skin sensitiser, DEREK NEXUS contains an expert-derived functionality to provide negative predictions for skin sensitization. This functionality further evaluates those compounds which do not fire any skin sensitisation alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitisation data, producing the following outcomes:
- In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: inactive.
- For those query compounds where features in the molecule are found in non-alerting skin sensitisers in the Lhasa reference set, the prediction remains negative and the misclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
- In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the 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) using the following equation:
EC3Q = MWQ /(Σ ωNN / Σ TNN)
MW = molecular weight
T = Tanimoto similarity score
ω = weighting factor = (MWNN/EC3) x TNN
Q = query compound
NN = nearest neighbour
The EC3 is the estimated concentration needed to produce a stimulation index of 3.

5. APPLICABILITY DOMAIN
i. descriptor domain: if a substance activates an alert describing a structure activity relationship for skin sensitisation it can be considered to be within the applicablility domain. The aplicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound dose not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of a negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
ii. structural fragment domain: for skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, “no alerts fired” may be extrapolated to a negative prediction. However, the substructure I-O could also not be found in the Lhasa skin sensitisation negative prediction dataset. Therefore, this prediction should be considered with caution.
iii. mechanism domain: as the prediction is “no alerts fired” none of the mechanisms for skin sensitisation is predicted to be applicable to this structure.
iv. metabolic domain: not relevant.

6. ADEQUACY OF THE RESULT
- The uncertainty of the prediction
The structure did not match any structural alerts or examples for skin sensitisation in DEREK, and the substructure I-O could not be found in the Lhasa skin sensitisation negative prediction dataset. Therefore, this prediction should be considered with caution.

The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments. The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The potential for skin sensitisation of the test material was predicted with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

GLP compliance:

no

Remarks:

calculation method

Type of study:

other: calculation method using the in silico model DEREK NEXUS

Justification for non-LLNA method:

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. It has been found to provide valuable in silico predictions on skin sensitisation.

Specific details on test material used for the study:

- Molecular weight: 549.9866
- SMILES: C1=C(C(=CC(=C1C2=CC(=C(C=C2C)OI)C(C)C)C)OI)C(C)C

Key result

Remarks on result:

other: The test material is predicted to be not sensitising to the skin

DEREK NEXUS version 6.0.1 did not match the query structure with any structural alerts or examples for skin sensitisation. However, the query structure also contains features that were not found in the Lhasa skin sensitisation negative prediction dataset (unclassified). The test material is predicted to be not sensitising to the skin, but this prediction should be considered with care.

Interpretation of results:

other: Not sensitising according to EU criteria

Conclusions:

DEREK NEXUS version 6.0.1 did not match the query structure with any structural alerts or examples for skin sensitisation. However, the query structure also contains features that were not found in the Lhasa skin sensitisation negative prediction dataset (unclassified). The test material is predicted to be not sensitising to the skin, but this prediction should be considered with care.

Executive summary:

The potential for skin sensitisation of the test material was predicted with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 did not match the query structure with any structural alerts or examples for skin sensitisation. However, the query structure also contains features that were not found in the Lhasa skin sensitisation negative prediction dataset (unclassified). The test material is predicted to be not sensitising to the skin, but this prediction should be considered with care.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2018 to 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

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 sensitisation tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Details on the study design:

DOSE FORMULATION

Preparation of Test Material Stock, Spiking and Working Solutions
- No correction was made for the composition/purity of the test material.
- A solubility test was performed. First a test was performed in mM (not reported), however the properties of the test material need a solubility test in mg/mL. The test material was suspended in DMSO to a final concentration of 40 mg/mL (orange). The stock was sonicated (Time: 11 min; Temp.: 22.5 - 29.0 °C). The 100-fold dilution of the 40 and 20 mg/mL DMSO stock formed a non-homogeneous suspension. The 100-fold dilution of the 10 mg/mL stock formed a homogenous suspension (slight precipitation). The 100-fold dilution of the 5 and 2.5 mg/mL stock formed a clear solution. The 10 mg/mL concentration (final concentration 100 µg/mL) was selected as highest concentration for the main assay (limit of solubility).
- In the main experiments the test material was suspended in dimethyl sulfoxide (DMSO) at 10 mg/mL (hazy yellow to yellow suspension). The stock was sonicated (first experiment: Time: 11 min; Temp.: 28 - 33 °C, second experiment: Time: 10 min; Temp.: 25 - 31 °C). 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 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098 and 0.049 μg/mL (final concentration DMSO of 1%). All concentrations of the test material were tested in triplicate. At concentrations of 5 mg/mL and higher the test material formed a suspension in dimethyl sulfoxide whereas at 2.5 mg/mL and lower it was fully soluble.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test material concentrations were used within 3.5 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 for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted so that the final concentration of the positive control ranged 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) foetal 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) foetal 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) foetal 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 66 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.0 - 37.2 °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 these deviations are considered not to affect the study integrity.

Subculturing
- 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).

EXPERIMENTAL DESIGN

Plating of Cells
- For testing, cells were 80-90 % 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+17 in experiment 1 and P+10 in experiment 2.

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 materials 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. Initially, experiment 1 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.

Luciferase Activity Measurement
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilised) from Promega (Leiden, The Netherlands) 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 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) 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.

ACCEPTABILITY CRITERIA
The KeratinoSens™ test is considered acceptable if it meets the following criteria:
- 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).
- 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.
- 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.


INTERPRETATION

Data analysis
The following parameters are calculated in the KeratinoSens™ test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- 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
- The IC50 and IC30 concentration values for 50 and 30 % reduction of cellular viability.
Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction = (Lsample – Lblank) / (Lvehicle – Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lvehicle is the average luminescence reading in the wells containing cells and vehicle (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.
Equation 2: EC1.5 = (Cb – Ca) x [(1.5 – Ia) / (Ib – Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = [(Vsample – Vblank) / (Vvehicle – Vblank)] x 100
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vvehicle is the average MTT-absorbance reading in the wells containing cells and vehicle (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
Equation 4: ICx = (Cb – Ca) x [((100 – x) – Va) / (Vb – Va)] + Ca
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability
Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability
Va is the % viability at the lowest concentration with > x% reduction in viability
Vb is the % viability at the highest concentration with < x% reduction in viability
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:
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.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.53 and the EC1.5 was 109 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.28 and the EC1.5 was 88 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (µM)

Value:

63

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (µM)

Value:

24

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

2.56

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

2.13

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
- The test material 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. The results of the positive control are summarised in Table 2. An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3.
- Two independent experiments were performed. The cells were in these experiments incubated with the test material in a concentration range of 0.05 – 100 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 material 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.
- A dose related luminescence activity induction was observed after treatment with the test material. The Imax was 2.56 and the EC1.5 was 63 µg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.53 and the EC1.5 was 109 µM.

Experiment 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material 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.
- A dose related luminescence activity induction was observed after treatment with the test material. The Imax was 2.13 and the EC1.5 was 24 µg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.28 and the EC1.5 was 88 µM.

Both tests 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 (109 µM and 88 µM in experiment 1 and 2, respectively). A clear dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in in both experiments (2.53-fold and 2.28-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 and 8.8 % in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
- The test material showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 63 and 24 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.56-fold and 2.13-fold in experiment 1 and 2 respectively. The test material 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.

Table 1: Overview Luminescence Induction and Cell Viability of The Test Material in Experiment 1 and 2

Concentration (µg/mL)	0.049	0.098	0.20	0.39	0.78	1.6	3.1	6.3	13	25	50	100
Exp 1 luminescence	0.84	1.04	1.01	1.09	1.10	1.21	1.12	1.10	1.08	1.41	1.14	2.56**
Exp 1 viability (%)	100.8	98.7	102.5	97.3	98.3	98.7	102.7	103.8	111.3	120.3	132.8	133.9
Exp 2 luminescence	1.05	1.06	1.02	1.08	1.00	1.09	1.16	1.24	1.37	1.51^***	1.90^***	2.13^***
Exp 2 viability (%)	152.0	129.0	124.4	119.8	125.8	128.2	145.0	164.4	185.9	206.8	194.3	159.4

^**p < 0.01 Student’s t test,^***p < 0.001 Student’s t test

 

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	0.79	0.87	1.11	1.14	1.63^***	^2.53***
Exp 1 viability (%)	104.2	105.8	103.8	111.2	113.9	113.6
Exp 2 luminescence	1.08	1.24	1.23	1.32	1.77^***	2.28^**
Exp 2 viability (%)	105.7	100.1	105.1	110.6	105.5	83.8

^**p<0.01 Student’s t test,^***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 Material Experiment 1	63	2.56	NA	NA
Test Material Experiment 2	24	2.13	NA	NA
Pos Control Experiment 1	109	2.53	NA	NA
Pos Control Experiment 2	88	2.28	NA	NA

NA = Not applicable

Interpretation of results:

other: Positive in the Keratinosens assay

Conclusions:

Under the conditions of this study, the test material 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.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 422D, under GLP conditions.

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The test material was suspended in dimethyl sulfoxide at 10 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 0.05 – 100 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: 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 (109 and 88 µM in experiment 1 and 2, respectively). A clear dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in both experiments (2.53 -fold and 2.28-fold in experiment 1 and experiment 2, respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (9.0 and 8.8 % in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test material showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 63 µg/mL and 24 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.56-fold and 2.13-fold in experiment 1 and 2 respectively. The test material 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.

Under the conditions of this study, the test material is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2018 to 06 August 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: ca 11 weeks old
- Weight at study initiation: 21.0 - 23.8 g
- Housing: animals were housed in groups of 5 in polycarbonate cages containing sterilized sawdust as bedding material. For psychological/environmental enrichment, animals were provided with paper and shelters.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 40 - 68 %
- Air changes: ten or more air changes per hour
- Photoperiod: 12 hours dark / 12 hours light

Vehicle:

dimethylformamide

Concentration:

0 (vehicle control), 1, 5, 10 % (w/w)

No. of animals per dose:

5 females per dose

Details on study design:

PRE-SCREEN TESTS:
Initially, two test material concentrations were tested; a 25 % and 50 % concentration.
Based on the results of the initially treated animals, six additional animals were treated in a similar manner with three lower concentrations (2 %, 5 % and 10 %) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labelled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY
Three groups of five animals were treated with one test material concentration per group. The highest test material concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

- Induction: Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test material.

- Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

- Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.

- Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In-life Procedures, Observations, and Measurements
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Clinical observations: Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
- Body weights: Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).
- Irritation: Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing), according to the Draize system.
- Ear thickness measurements: Ear thickness measurements were performed for all animals prior to dosing on Days 1 and 3, and on Day 6 using a digital thickness gauge (Kroeplin C110T-K).
- Terminal procedures: No necropsy was performed, since all animals survived until the end of the observation period.

ANALYSIS
A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitiser.

Positive control substance(s):

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

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the Test Facility. In this study, performed in June 2018, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Alpha- Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 11 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”.
The SI values calculated for the positive control item concentrations 5, 10 and 25 % were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2 %.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Value:

0.7

Test group / Remarks:

1 % (w/w)

Parameter:

SI

Value:

1.9

Test group / Remarks:

5 % (w/w)

Parameter:

SI

Value:

4.2

Test group / Remarks:

10 % (w/w)

Cellular proliferation data / Observations:

PRE-SCREEN TESTS
At a 25 % and 50 % test material concentration, variation in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria.
At a 2 %, 5 % and 10 % test material concentration, no signs of systemic toxicity were noted, only very slight erythema and scaliness was observed. Ear thickness measurements were slightly exceeding the 25 % threshold for one ear per animal on Day 6. A 1 %, 5 % and 10 % concentration were selected as the concentrations to be used in the main study to test a wider range of concentrations. Ear thickness measurements were added to the main study, since the results of the ear thickness measurements were not conclusive.

MAIN STUDY
- Skin reactions / Irritation: The very slight erythema, scaliness and/or bald skin behind the ears and head as shown by the animals treated at 5 % and 10 % between Days 2 and 6 were considered not to have a toxicologically significant effect on the activity of the nodes.

- Ear thickness: Variation in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values for the vehicle control animals, the animals treated at 1 % and four animals treated at 5 %. The variation slightly exceeded 25 % for all animals treated at 10 % and one animal treated at 5 %. As no correlation was seen between individual ear thickness measurement and DPM values, this slight increase above the selection threshold was considered not to have affected the results of the study.

- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

- Macroscopic Examination of the Lymph Nodes and Surrounding Area: The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals treated at 10 %, which were considered to be enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

- Radioactivity Measurements and SI Values: Mean DPM/animal values for the experimental groups treated with test material concentrations 1, 5 and 10 % were 749, 2144 and 4747 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1133 DPM. The SI values calculated for the test material concentrations 1, 5 and 10 % were 0.7, 1.9 and 4.2, respectively.

Table 1: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI) in the Main Study

Test material (% w/w)	Animal	Size nodes†		DPM/ animal	mean DPM ± SEM	mean SI ± SEM
		left	right
0 (vehicle control)	1	n	n	1319	1133 ± 137	1.0 ± 0.1
	2	n	n	832
	3	n	n	1210
	4	n	n	803
	5	n	n	1499
1	6	n	n	666	749 ± 49	0.7 ± 0.0
	7	n	n	783
	8	n	n	625
	9	n	n	907
	10	n	n	762
5	11	n	n	2655	2144 ± 313	1.9 ± 0.3
	12	n	n	2156
	13	n	n	1287
	14	n	n	1636
	15	n	n	2986
10	16	+	+	3593	4747 ± 658	4.2 ± 0.6
	17	+	+	5851
	18	+	+	4507
	19	+	+	3159
	20	+	+	6625

 †  Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal)

DPM = Disintegrations per minute.

SEM = Standard Error of the Mean.

Interpretation of results:

other: Skin sensitiser according to EU criteria

Conclusions:

The results from the study indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 7.4 % was calculated.
The six-month reliability check with the positive control indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.
Based on these results it should be considered to regard the test material as a skin sensitiser.

Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600, under GLP conditions.

Test material concentrations selected for the main study were based on the results of a pre-screen test. At a 25 % and 50 % test material concentration, variation in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 2 %, 5 % and 10 % test material concentration, no signs of systemic toxicity were noted, only very slight erythema and scaliness was observed. Ear thickness measurements were slightly exceeding the 25 % threshold for one ear per animal on Day 6. A 1 %, 5 % and 10 % concentration were selected as the concentrations to be used in the main study to test a wider range of concentrations. Ear thickness measurements were added to the main study, since the results of the ear thickness measurements were not conclusive.

In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 1, 5 or 10 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide). 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.

Variation in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values for the vehicle control animals, the animals treated at 1 % and four animals treated at 5%. The variation slightly exceeded 25 % for all animals treated at 10 % and one animal treated at 5 %. As no correlation was seen between individual ear thickness measurement and DPM values, this slight increase above the selection threshold was considered not to have affected the results of the study.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals treated at 10 %, which were considered to be enlarged. 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 material concentrations 1, 5 and 10 % were 749, 2144 and 4747 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1133 DPM. The SI values calculated for the test material concentrations 1, 5 and 10 % were 0.7, 1.9 and 4.2, respectively.

These results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 7.4 % was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

Based on these results it should be considered to regard the test material as a skin sensitiser.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2018 to 16 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: Direct Peptide Reactivity Assay

Details on the study design:

Skin sensitisation (In chemico test system)

Dose Formulation and Analysis
- Preparation of Test Material
No correction for the purity/composition of the test material was performed.
Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol and ethanol.
Any residual volumes were discarded.

At a concentration of 100 mM, the test material was not soluble in ACN, MQ, ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), methanol and ethanol.
Therefore, no DPRA study could be performed and the study was stopped.

Positive control results:

Not applicable - study terminated.

Remarks on result:

not measured/tested

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since no suitable solvent compatible with the DPRA to dissolve the test material at the desired concentration could be found, no main experiment could be performed.
Therefore, the reactivity of the test material towards SPCC and SPCL could not be determined.

Executive summary:

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). However, since no suitable solvent also compatible with the Direct Peptide Reactivity Assay (DPRA) could be identified to dissolve the test item at the desired concentration, the reactivity of the test material towards SPCC or SPCL could not be determined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro- Keratinosens Assay (Woutersen, 2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 422D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

During the studyt the test material was suspended in dimethyl sulfoxide at 10 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 0.05 – 100 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: 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 (109 and 88 µM in experiment 1 and 2, respectively). A clear dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in both experiments (2.53 -fold and 2.28-fold in experiment 1 and experiment 2, respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (9.0 and 8.8 % in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test material showed no toxicity (no IC30 and IC50 value). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 63 µg/mL and 24 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 2.56-fold and 2.13-fold in experiment 1 and 2 respectively. The test material 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.

Under the conditions of this study, the test material is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

In silico - DEREK calculation

The potential for skin sensitisation of the test material was predicted with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS version 6.0.1 did not match the query structure with any structural alerts or examples for skin sensitisation. However, the query structure also contains features that were not found in the Lhasa skin sensitisation negative prediction dataset (unclassified). Thymol iodide is predicted to be not sensitising to the skin, but this prediction should be considered with care.

In Chemico, Direct Peptide Reactivity Assay (Reinen, 2018)

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). However, since no suitable solvent also compatible with the Direct Peptide Reactivity Assay (DPRA) could be identified to dissolve the test item at the desired concentration, the reactivity of the test material towards SPCC or SPCL could not be determined.

Weight of Evidence (Pelgrom, 2018)

The objective of this study was to reach an overall conclusion on the endpoint skin sensitisation based on all available relevant information, including alternative testing data.

A DEREK assessment, DPRA assay and a 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.

A reliable DPRA study could not be performed, due to solubility issues with the substance under the experimental conditions of the test. The positive result observed in the KeratinoSens™ assay is indicative for skin sensitising potential of the substance.

Based on these results, no overall conclusion on skin sensitising potential can be drawn, as no individual in vitro test is considered reliable to predict skin sensitising properties of a substance. Based on a weight of evidence from the studies performed and the lack of conclusive information on skin sensitising potential and/or potency, further testing is needed. Performing another in vitro test (STEP 2, U-SENS™) will not result in a final conclusion on skin sensitisation, as both a negative and a positive result of this test would need to be followed by the LLNA (in the absence of a reliable and acceptable in vitro skin sensitisation test that may determine the skin sensitising potency).

In vivo Local Lymph Node Assay (van Sas, 2018)

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Test material concentrations selected for the main study were based on the results of a pre-screen test. At a 25 % and 50 % test material concentration, variation in ear thickness during the observation period were more than 25 % from Day 1 pre-dose values and/or clinical signs of systemic toxicity were noted. Therefore these concentrations did not meet the selection criteria. At a 2 %, 5 % and 10 % test material concentration, no signs of systemic toxicity were noted, only very slight erythema and scaliness was observed. Ear thickness measurements were slightly exceeding the 25 % threshold for one ear per animal on Day 6. A 1 %, 5 % and 10 % concentration were selected as the concentrations to be used in the main study to test a wider range of concentrations. Ear thickness measurements were added to the main study, since the results of the ear thickness measurements were not conclusive.

In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 1, 5 or 10 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-dimethylformamide). 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.

Variation in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values for the vehicle control animals, the animals treated at 1 % and four animals treated at 5 %. The variation slightly exceeded 25 % for all animals treated at 10 % and one animal treated at 5 %. As no correlation was seen between individual ear thickness measurement and DPM values, this slight increase above the selection threshold was considered not to have affected the results of the study.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in the animals treated at 10%, which were considered to be enlarged. 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 material concentrations 1, 5 and 10 % were 749, 2144 and 4747 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1133 DPM. The SI values calculated for the test material concentrations 1, 5 and 10 % were 0.7, 1.9 and 4.2, respectively.

These results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI =3) of 7.4 % was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

Based on these results it should be considered to regard the test material as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the Regulation (EC) No. 1272/2008, the substance requires classification as a skin sensitiser (Category 1B) and is assigned the hazard statement H317: May cause an allergic skin reaction.