Registration Dossier

Administrative data

Description of key information

Weight of Evidence: sensitising, 2019

1. Molecular initiating Key Event 1: negative (no or minimal peptide reactivity), DPRA, OECD TG 442C, 2017

2. Molecular initiating Key Event 2: negative (no LC induction > 1.5 in 2 of 3 replicates), KeratinoSens, OECD TG 442D, 2017

3. Molecular initiating Key Event 3: positive (three of four independent runs positive of four with EC150 ≥ 3.0 and ≤ 24 μg/mL), U-SENS, OECD TG 442E, 2018

4. LLNA: Skin sensitisation: sensitising (EC3 = 28.8%), female mice, OECD TG 429, 2002

Note: the applicant cannot exclude potential for false-positive response in aforementioned assays.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
Remarks:
Additional investigations for dimerisation potential were conducted.
GLP compliance:
no
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the [name redacted] testing facility’.
- Acetonitrile (i.e. the standard solvent of the SOP) was used for preparation of test solutions. The test was run according to the final validated SOP published by ECVAM (DB-ALM protocol 154). Reference: ECVAM, Direct peptide reactivity assay (DPRA) for skin sensitization testing DB-ALM database (2014).
- HPLC-UV and LC-MS (Dimerization investigation) methodology are reported in the full study report.
- Acceptability criteria:
(i) Cys-peptide depletion standard deviation should be < 14.9% (Test item: Actual = 0.8%)
(ii) Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 0.1%)
(iii) The co-elution controls indicated no co-elution with an UV-absorbing component.
(iv) Calibration curves for the HPLC-UV analysis were as follows: (i) Cysteine r2 = 0.9922 and (ii) Lysine r2 = 1.0000
(v) Mean peptide concentrations in reference item replicate control C: was 0.5 +/- 0.05 mM (actual = 0.50 to 0.50 mM in acetonitrile)
(vi) CoV in reference items B and C in acetonitrile: 6.42% (Cys peptide) and 0.34% (Lys peptide)

(vii) Positive Control: cinnamic aldehyde:
- Cysteine peptide: Should be between 60.8% and 100%
Actual = 65.4% (SD = 1.3%) and CoV = 2.0%
Cys-peptide depletion standard deviation should be < 14.9% (PC : Actual = 1.3%)
CoV should be < 15.0% (PC : Actual = 2.0%)
- Lysine peptide Should be between 40.2% and 69.0%
Actual = 43.5% (SD = 1.7%) and CoV = 3.9%
Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 1.7%)
CoV should be < 15.0% (PC : Actual = 3.9%)
All acceptance criteria were fulfilled for the PC
(viii) Mean peptide concentrations in reference item replicate control A: was 0.5 +/- 0.05 mM (actual = 0.49 to 0.51 mM in acetonitrile)

(ix) Stability of vehicle control: A control experiment according to the DB-ALM protocol 154 to test whether Acetonitrile has no effect on peptide stability was performed. A 0.5mM Cysteine solution was incubated for 24 h in Phosphate buffer / acetonitrile, and subsequently injected every 3 h for 48 h. The CV of these samples was found to be 3.4% (DB-ALM protocol requirement: < 15%).

All acceptability criteria were met.

- Peptide dimerization: were monitored by LC-MS allowing quantification of the peptide dimers. Correct identification of the peptide dimer molecular formula was possible based on calculated and observed exact mass and comparison to synthetic references.
Reference: OECD TG 442C paragraph 23: 23. There may be test test items which could promote the oxidation of the cysteine peptide. The peak of the dimerised cysteine peptide may be visually monitored. If dimerization appears to have occurred, this should be noted as percent peptide depletion may be over-estimated leading to false positive predictions and/or assignment to a higher reactivity class.

- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.1) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.4) – full details on source provided in full study report.
Cysteine-dimer peptide: (MW=1499.2) – full details on source provided in full study report.

Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; > 99% food-grade purity)
Negative control (NC): Vehicle = acetonitrile

Test item preparation:
The test substance was prepared as a 100 mM stock solution in acetonitrile prior to incubation with synthetic peptides. The test item was freely soluble in acetonitrile at 100 mM. Three replicates of the test item were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (NC) were incubated with the peptides. The peptide depletion of test item incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test item the mean peptide depletion as average of Cys and Lys peptide depletion is calculated and used for evaluation of the test item reactivity.

Dimerisation investigation:
In parallel, test item, PC and NC samples were 100-fold diluted and analysed with high resolution LC-MS were conducted. In this parallel analysis, peptide depletion, dimer formation and adduct formation were separately investigated, both for the test item and the control cinnamic aldehyde

Evaluation of results:
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity
> 22.62 < 42.47 moderate reactivity
> 6.38 < 22.62 low reactivity
< 6.38 minimal reactivity

Dimerization investigation:
With completed LC-MS analysis, formation of peptide dimer and possible adducts is screened in addition to peptide depletion. If depletion of the Cys-peptide is observed AND the following two conditions are met:
a) Significantly enhanced formation of peptide Dimer or other peptide oxidation products such as the sulfoxide in test samples as compared to control reference samples
b) Lack of formation of a direct peptide adduct
Then there is no evidence for a direct reactivity, and observed peptide depletion can be explained by the chemical-catalysed peptide oxidation rather than direct reactivity. In this case, the scientifically most appropriate conclusion is that the test does not indicate reactivity of the test chemical.

However, if both peptide oxidation and adduct formation occur in parallel, then certainly chemicals are still rated as positive and peptide reactive.

This prediction model is not part of the validated protocol, but it takes into account a well described shortcoming of the DPRA. Since it does not form part of the validated protocol, this additional analysis should be used in a weight-of-evidence based analysis of the sensitization potential of a test chemical.
Positive control results:
- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 64.4% (high reactivity)
- PC LYS-peptide depletion (mean): 43.5% (moderate reactivity)
- The positive control has a theoretical Michael acceptor structural alert for reactivity with a cysteine containing peptide, and the predicted direct peptide adduct is shown (within the full study report) with a theoretical exact mass of 882.4058. Therefore the [M+H]+ ion of 883.4131 was searched for by LC-MS. A new peak eluting at 9.34 min was observed and identified as [M+H]+ ion. The MS analysis indicated that the measured exact mass at this retention time is completely in line with the predicted mass which confirms that this peak is a direct adduct between the positive control and the test peptide, confirming that the positive control has direct peptide reactivity.
Parameter:
other: Cys-peptide depletion
Run / experiment:
Mean (%)
Value:
29.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Applicant indicates: NO or MINIMAL REACTIVITY CLASS
Remarks:
See 'any other information on results incl. tables' for dimerization investigations
Parameter:
other: Lys-peptide depletion
Run / experiment:
Mean (%)
Value:
0.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the [name redacted] testing facility’.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Acceptability criteria: All acceptability criteria were met.
(i) Cys-peptide depletion standard deviation should be < 14.9% (Test item: Actual = 0.8%)
(ii) Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 0.1%)
(iii) The co-elution controls indicated no co-elution with an UV-absorbing component.
(iv) Calibration curves for the HPLC-UV analysis were as follows: (i) Cysteine r2 = 0.9922 and (ii) Lysine r2 = 1.0000
(v) Mean peptide concentrations in reference item replicate control C: was 0.5 +/- 0.05 mM (actual = 0.50 to 0.50 mM in acetonitrile)
(vi) CoV in reference items B and C in acetonitrile: 6.42% (Cys peptide) and 0.34% (Lys peptide)

(vii) Positive Control: cinnamic aldehyde:
- Cysteine peptide: Should be between 60.8% and 100%
Actual = 65.4% (SD = 1.3%) and CoV = 2.0%
Cys-peptide depletion standard deviation should be < 14.9% (PC : Actual = 1.3%)
CoV should be < 15.0% (PC : Actual = 2.0%)
- Lysine peptide Should be between 40.2% and 69.0%
Actual = 43.5% (SD = 1.7%) and CoV = 3.9%
Lys-peptide depletion standard deviation < 11.6%. (Test item: Actual = 1.7%)
CoV should be < 15.0% (PC : Actual = 3.9%)
All acceptance criteria were fulfilled for the PC
(viii) Mean peptide concentrations in reference item replicate control A: was 0.5 +/- 0.05 mM (actual = 0.49 to 0.51 mM in acetonitrile)

(ix) Stability of vehicle control: A control experiment according to the DB-ALM protocol 154 to test whether Acetonitrile has no effect on peptide stability was performed. A 0.5mM Cysteine solution was incubated for 24 h in Phosphate buffer / acetonitrile, and subsequently injected every 3 h for 48 h. The CV of these samples was found to be 3.4% (DB-ALM protocol requirement: < 15%).

Dimerisation investigation:

1. Peptide adduct investigation:

LC-MS allowed quantification of the parent peptide as [M+H]+ion eluting at a retention time of 7.16 min. Correct identification of the parent peptide molecular formula was possible based on calculated and observed exact mass.This peak was integrated for all samples and used to calculated peptide depletion as done for HPLC-UV analysis. The test item gave 28.4% depletion of the Cys-peptide when measured with the LC-MS method. This value is close to the value (29.9%) reported using HPLC-UV, and thus peptide quantification with this alternative method is reliable (also consistent with the positive control results), and if using the depletion only, this alternative analysis method leads to identical conclusions.

2. Peptide dimerization LC-MS

LC-MS allowed quantification of the peptide dimer, which is observed as the double charged [M+2H]2+ ion eluting at a retention time of 8.17 minutes. Correct identification of the peptide dimer molecular formula was possible based on calculated and observed exact mass and comparison to synthetic reference. For each sample, this peak was integrated and quantified based on a calibration curve. This peak was enhanced in all three replicate samples of the test substance. Compared to Reference C, the parent peptide was reduced by 0.14 mM in the test samples, while the dimer peak in the test samples compared to the control samples was enhanced by 0.077mM corresponding to 0.15 mM test peptide. Quantitatively, the formed dimer can thus fully account for the peptide loss.

3. Expert Judgement assessment:

The test item does not have a typical structural alert for peptide adduct formation. Theoretically, the only presumed mode of action would be an acyl transfer, whereby the lactone is transformed to a thioester, although this process is energetically normally not favoured. Therefore the [M+H]+ ion of 1003.5695 was searched for by LC-MS. No new peak with this mass was observed. The test item did not clearly form a direct acyl transfer peptide adduct or similar direct adducts with the theoretically calculated MW 1002.6.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test item is not considered to be potentially sensitising to the skin.
Executive summary:

The study was performed to the validated OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA). The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. All test acceptability criteria were met. The test item was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by the test item was determined by HPLC-UV. In parallel, the same samples were 100-fold diluted and analysed with high resolution LC-MS. In this parallel analysis, peptide depletion, dimer formation and adduct formation were separately investigated, both for the test item and the control cinnamic aldehyde.When using HPLC-UV analysis only, the test item led to 29.9.1% depletion of the Cys-peptide and 0.1% depletion of the Lys-peptide. In extension to the assay LC-MS investigations of Cys-peptide oxidation / dimerization was conducted. When using HPLC-UV analysis only, the test item led to 29.9% depletion of the Cys-peptide. When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test item. At the same time the test item triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation / dimer formation rather than adduct formation / direct reactivity. No indication for a peptide adduct was found in this analysis.For the positive control (Cinnamic aldehyde), direct adduct formation was verified indicating direct reactivity for the positive control. Thus, based on this more sophisticated and detailed analysis, the test item is not directly peptide reactive, but can trigger peptide dimerization which is not considered itself as a molecular initiating event in skin sensitization.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
no
Remarks:
Method equivalent or similar to guideline, non-GLP. Well documented study in accordance with scientific principles and sufficient for assessment.
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422D – In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘KeratinoSens assay: Proficiency testing at the testing facility’.
- DMSO (i.e. the standard solvent of the SOP) could be used for preparation of test solutions. The test was run according to the final validated SOP published by ECVAM (DB-ALM protocol 155). Reference: ECVAM, KeratinoSens protocol for skin sensitization testing DB-ALM database, (2014).

- Acceptability criteria:
All acceptability criteria were met.
(i) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be above 1.5: Actual PC: replicate 1: 14.17 ; replicate 2: 15.43 and replicate 3: 17.53 : mean: 15.71 and the results were statistically significant. The acceptability criteria was met.
(ii) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be within 2 standard deviations of the HCD (2 to 8) and EC1.5 should be between 7 μM and 30 μM. The acceptability criteria was met.
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual replicate 1: 14.10% ; replicate 2: 11.34% and replicate 3: 10.17%. The acceptability criteria was met.

Cell line used:
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene AKR1C2. The cell line was developed by the testing lab and stored on liquid nitrogen. It was grown in 10 cm petri dishes as described in the SOP to 80% confluency prior to testing for 3 – 4 days. Cells were counted using a counting chamber and adjusted to the desired density. During seeding into 96-well plates, the cell suspension was gently stirred and cell sedimentation was avoided by repeatedly pipetting up and down to ensure homogeneous distribution of cells.

Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Preparation of test chemicals:
Test item and/or reference item (PC or VC) were dissolved in DMSO at a concentration of 200 mM and serially diluted in DMSO to obtain 12 final concentrations ranging from 0.98 μM to 2000 μM. The concentration was limited to between 1.0 and 100 μM based on cytotoxicity of the test item to the cell line. < 70% viability was observed at concentrations of around 100 to 150 μM.

Endpoint & Endpoint Detection:
Two endpoints are measured:
(i) Luciferase induction after a 48 h treatment with test substances and
(ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell batch and made up with the same dilutions of the test chemicals
Luminescence was read in a Luminometer programmed to
i. add 50 μl of the luciferase substrate to each well,
ii. to then wait for 1 second and
iii. then to integrate the luciferase activity for 2 seconds.

Cell viability assay MTT:
Test item IC50: IC50 value as the concentration in μM reducing the viability by 50%
Replicate 1: 102.94 μM ; replicate 2: 109.32 μM and replicate 3: 605.27 μM : mean: 189.56 μM ; SD = 288.2

Luciferase assay
Imax indicating maximum fold-induction up to concentration 1000 μM
Replicate 1: 1.36 ; replicate 2: 1.13 and replicate 3: 1.51 : mean: 1.33 ; SD = 0.19

Determinations:
EC1.5
Replicate 1: N.I. ; replicate 2: N.I. and replicate 3: 13.12 μM : mean: N.I. ; SD = -
EC2
Replicate 1: N.I. ; replicate 2: N.I. and replicate 3: N.I. : mean: N.I. ; SD = -
EC3
Replicate 1: N.I. ; replicate 2: N.I. and replicate 3: N.I. : mean: N.I. ; SD = -
Where: N.I. = no induction above given threshold

Evaluation criteria
Test item considered ‘positive’ where
1. The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above the solvent control in a repetition as determined by students T-test.
The EC1.5 value is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to 1.5, the substance is still rated negative and no EC1.5 value is calculated)
2. At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining value), the cellular viability is above 70%.
3. There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

Results:
1 out of 3 positive replicates
Overall conclusion negative. In two of three repetitions, no induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is thus rated as a non-sensitizer. This conclusion is also clearly supported by the analysis of the dose-response curve, with overall no induction of the luciferase reporter gene to be observed.
Positive control results:
All acceptability criteria were met.
(i) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be above 1.5: Actual PC: replicate 1: 14.17 ; replicate 2: 15.43 and replicate 3: 17.53 : mean: 15.71 and the results were statistically significant. The acceptability criteria was met.
(ii) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be within 2 standard deviations of the HCD (2 to 8) and EC1.5 should be between 7 μM and 30 μM. The acceptability criteria was met.

Historical results for luciferase induction by the positive control in the test laboratory: average and standard deviations from 242 valid runs conducted in years 2014 through 2016 are presented in the full study report.
Parameter:
other: EC1.5
Run / experiment:
mean (n = 3)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: - The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442D. The results of the testing on the proficiency chemicals at the test facility is described in a separate test report (full references provided in the full study report): ‘KeratinoSens assay: Proficiency testing at the testing facility’.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable. Three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8.

- Acceptability criteria:
All acceptability criteria were met.
(i) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be above 1.5: Actual PC: replicate 1: 14.17 ; replicate 2: 15.43 and replicate 3: 17.53 : mean: 15.71 and the results were statistically significant. The acceptability criteria was met.
(ii) luciferase activity induction: 64 μM EC1.5 with PC (cinnamic aldehyde) should be within 2 standard deviations of the HCD (2 to 8) and EC1.5 should be between 7 μM and 30 μM. The acceptability criteria was met.
(iii) average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition: the % variability in solvent controls: Actual replicate 1: 14.10% ; replicate 2: 11.34% and replicate 3: 10.17%. The acceptability criteria was met.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test item is not considered to be potentially sensitising to the skin.
Executive summary:

The study was performed to the validated OECD TG 442D in vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method.The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. All test acceptability criteria were met. The test item was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSens assay at 12 concentrations in three repetitions, each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.The test item was weakly cytotoxic to the KeratinoSens cells. In two out of three repetitions, it did not induce the luciferase gene above a threshold of 1.5.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline Study performed under GLP. Applicant assessment indicates that there are test item specific properties that confound the assay (methodological deficiencies) and therefore exclusion of false-positive. Study is incorporated on precautionary basis.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Absence of preliminary range finder and no documentation of dose range selection. Absence of assessment of ear thickness measurements.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: April 2002; signature: May 2002
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 16.7 to 19.4 g
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding

- Diet (ad libitum): Pelleted standard Kliba 3433, batch no. 67102 mouse maintenance diet (Recognised Supplier); ad libitum
- Water (ad libitum): mains tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: Fro: 05-11-2002 To: 05-11-2002
Vehicle:
other: ethanol : water (7:3 v/v)
Concentration:
- Definitive test: 0 (control), 0.1%, 1%, 10% and 100% w/v
No. of animals per dose:
Main test: 4 females per dose group
Details on study design:
RANGE FINDING TESTS:
No preliminary testing was conducted within the study. The choice of the dose range employed was not clearly documented within the study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser". The data was to be compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item at concentrations of 0.1%, 1%, 10% and 100% w/v in ethanol : water (7:3 v/v). A further group of five mice received the vehicle alone in the same manner. The application volume, 25 μl, was spread over the entire dorsal surface (ca. 8 mm) of each ear lobe once daily for three consecutive days.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl 3H-methyl thymidine (3HTdR:83 μCi/ml) giving a total of 20.3 μCi to each mouse.

After treatment. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 IJm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 oc for 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a 13-scintillation counter. Similarly, background 3HTdR levels were also measured

Observations:
- Clinical Observations: All animals were observed daily on Days 1 to 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded prior to acclimitisation and Day 6 (prior to termination).
- Ear thickness measurements: Not determined durign the test. Observations and scoring of swelling of ears was completed only. Applicant assessment indicates: The grading system was not clearly indicated.

Determination of 3HTdR Incorporation:
After treatment. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 degrees Celsius for 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a 13-scintillation counter. Similarly, background 3HTdR levels were also measurede
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship.
Positive control results:
In a previously conducted non-concurrent 'positive control study' (details attached to the full study report) performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde at 5, 10 and 25% w/v in acetone/olive oil 4:1 vehicle. The highest concentration tested showed a Stimulation Index (SI) of 7.1 and met the criteria for a 'positive' result and EC3 = 11.3% (w/v).
The results of routine positive control testing (Historic Control Data) performed according to OECD TG 429, using α-hexylcinnamaldehyde indicated positive results.
Parameter:
SI
Value:
1.2
Test group / Remarks:
0.1% v/v test item in ethanol : water (7:3 v/v)
Parameter:
SI
Value:
1.1
Test group / Remarks:
1% v/v test item in ethanol : water (7:3 v/v)
Parameter:
SI
Value:
2.1
Test group / Remarks:
10% v/v test item in ethanol : water (7:3 v/v)
Parameter:
SI
Value:
6.4
Test group / Remarks:
100% test item

There were no significant test item related clinical observations. Moderated swelling of both dosing sites was observed, at one hour observation in all animals in the 100% test item group. The effect lasted for four days. One day after first application a slight erythema was noted at both dosing sites in all animals of 100% test item group. This erythema lasted for four days. There was no effect on body weight of administration of the test item (all bodyweights were within the range common to the strain and age).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is considered to be sensitising to the skin. Applicant assessment indicates there are methodological deficiencies within the test specifically, the choice of dose range was not adequately characterised within the study report and/or there was an absence of ear thickness measurements and/or indications of moderate ear swelling and/or slight erythema in the 100% test item group. Therefore false-positive cannot be excluded. Study is incorporated on precautionary basis.
Executive summary:

The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. The concentrations selected for the main test were 0% (control), 0.1%, 1%, 10% and 100% w/v within ethanol : water (7:3 v/v) vehicle as appropriate. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded prior to acclimatisation and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. There were no deaths or signs of systemic toxicity, and body weights were comparable to controls. Moderated swelling of both dosing sites was observed, at one hour observation in all animals in the 100% test item group. The effect lasted for four days. One day after first application a slight erythema was noted at both dosing sites in all animals of 100% test item group. This erythema lasted for four days. There was no effect on body weight of administration of the test item (all bodyweights were within the range common to the strain and age). A stimulation index (SI) was recorded for each concentration as follows: 0.1%w/v: SI = 1.2, 1%w/v: SI = 1.1 and 10%w/v: SI = 2.1 and 100%: SI = 6.4. The latter two concentrations were used to calculate an EC3 of 28.8%. Accordingly, the test material was considered to be sensitising under the conditions of the test. Applicant assessment indicates there are methodological deficiencies within the test specifically, the choice of dose range was not adequately characterised within the study report and/or there was an absence of ear thickness measurements and/or indications of moderate ear swelling and/or slight erythema in the 100% test item group. Which yielded the single positive result.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin Sensitisation:

1. Key Study – Molecular initiating Key Event 1: DPRA, OECD TG 442C, 2017: The study was performed to the validated OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA). The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. All test acceptability criteria were met. The test item was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by the test item was determined by HPLC-UV. In parallel, the same samples were 100-fold diluted and analysed with high resolution LC-MS. In this parallel analysis, peptide depletion, dimer formation and adduct formation were separately investigated, both for the test item and the control cinnamic aldehyde.When using HPLC-UV analysis only, the test item led to 29.9.1% depletion of the Cys-peptide and 0.1% depletion of the Lys-peptide. In extension to the assay LC-MS investigations of Cys-peptide oxidation / dimerization was conducted. When using HPLC-UV analysis only, the test item led to 29.9% depletion of the Cys-peptide. When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test item. At the same time the test item triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation / dimer formation rather than adduct formation / direct reactivity. No indication for a peptide adduct was found in this analysis.For the positive control (Cinnamic aldehyde), direct adduct formation was verified indicating direct reactivity for the positive control. Thus, based on this more sophisticated and detailed analysis, the test item is not directly peptide reactive, but can only trigger peptide dimerization which is not considered itself as a molecular initiating event in skin sensitization.

 

2. Key Study – Molecular initiating Key Event 2: KeratinoSens, OECD TG 442D, 2017: The study was performed to the validated OECD TG 442Din vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method.The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. All test acceptability criteria were met. The test item was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSens assay at 12 concentrations in three repetitions, each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.The test item was weakly cytotoxic to the KeratinoSens cells. In two out of three repetitions, it did not induce the luciferase gene above a threshold of 1.5.

 

3. Key Study - Molecular initating Key Event 3: U-SENS, OECD TG 442E, 2018: The study was performed to the OECD TG 442E (In Vitro Skin Sensitisation Assays addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation)U937 cell line activation Test (U-SENS™) test method under GLP.The testing laboratory has previously demonstrated technical proficiency requirements associated with the assay. The test item was dissolved in dimethyl sulfoxide at 50 mg/mL (experiment 1, 3 and 4) and 2.5 mg/mL (experiment 2). In the first experiment the stock was diluted to six test concentrations (1.0, 10, 20, 50, 100 and 200 μg/mL). In the second, third and fourth experiment, a narrower dose-response analysis was performed. No precipitate was observed at any dose level tested. Four independent experiments were performed. All acceptability criteria were met. At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in experiment 1 and 99% in experiment 2, 3 and 4). The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (99% in all experiments). The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments. The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments. At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments. No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls. In all experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. In the first experiment the test item showed toxicity (CV70 value of 4.0 μg/mL). An induction of CD86 activity (EC150 value 3.0 μg/mL) was measured only at test concentrations with cell viability < 70%. This led to an individual run conclusion of Positive based on cytotoxicity and colour interference. In the second experiment the test item showed no toxicity (No CV70 value) and no induction of the CD86 activity (No EC150 value) was measured. This led to an individual run conclusion of Negative, which is probably due to the lower highest test concentration of 10 μg/mL. Since the first and second experiment were not concordant and additional third experiment was performed with the same concentration as the first experiment. In the third experiment the test item showed no toxicity (No CV70 value). A biologically relevant, induction of the CD86 activity (EC150 value of 24 μg/mL) was measured. This led to an individual run conclusion of Positive. As the concentration series was similar to the first experiment, a final fourth experiment was performed with a different concentration series. In the fourth experiment the test item showed no toxicity (No CV70 value). A biologically relevant, induction of the CD86 activity (EC150 value of < 15 μg/mL) was measured. This led to an individual run conclusion of Positive. Under the conditions of this study, the test item is considered to be potentially sensitising to the skin and/or increase in the expression levels of CD86 cell surface marker in the U937 cell line. On the basis of positive results observed in 3 out of 4 experiments and induction was observed at test concentrations with a cell viability of >70% compared to the vehicle control in 2 out of 4 experiments.

 

Applicant assessment indicates that within the U-SENS assay there is potential for false positive from OECD TG 442E paragraph 4, that for some surfactants the U-SENS positive results should be considered with caution due to high false-positive results (3 of 7) seen in the supporting literature. There are unique properties of the test item which may yield trace levels of linear unsaturated-carboxylate equivalents of the macrocyclic lactone with surfactant properties. These type of constituents (non-ionic surfactants) may have confounding issues for U-SENS non-specific CD86 expression (see: Piroird et al.,(2015): Toxicology in vitro, 29: 901-916). On this basis the result is accepted on a precautionary basis although potential for false positive cannot be excluded.

 

4. Key Study: OECD TG 429, 2002: The study was performed to OECD TG 429 under GLP to assess the skin sensitisation potential of the test material in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. The concentrations selected for the main test were 0% (control), 0.1%, 1%, 10% and 100% w/v within ethanol : water (7:3 v/v) vehicle as appropriate. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded prior to acclimatisation and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. There were no deaths or signs of systemic toxicity, and body weights were comparable to controls. Moderated swelling of both dosing sites was observed, at one hour observation in all animals in the 100% test item group. The effect lasted for four days. One day after first application a slight erythema was noted at both dosing sites in all animals of 100% test item group. This erythema lasted for four days. There was no effect on body weight of administration of the test item (all bodyweights were within the range common to the strain and age). A stimulation index (SI) was recorded for each concentration as follows: 0.1%w/v: SI = 1.2, 1%w/v: SI = 1.1 and 10%w/v: SI = 2.1 and 100%: SI = 6.4. The latter two concentrations were used to calculate an EC3 of 28.8%. Accordingly, the test material was considered to be sensitising under the conditions of the test.

 

Applicant assessment indicates there are methodological deficiencies within the test specifically, the choice of dose range was not adequately characterised within the study report and/or there was an absence of ear thickness measurements and/or indications of moderate ear swelling and/or slight erythema in the 100% test item group. Which yielded the single positive result. The test is being incorporated into the weight of evidence conservatively. For the purposes of potency assessment in event of legitimate sensitising properties.

 

Weight of Evidence Conclusion:

The applicant assesses by expert judgement that GHS skin sensitisation category 1B will be applied on a precautionary basis. The weight of evidence that there is no evidence of skin sensitisation in two validated in chemico (DPRA, eq. to OECD TG 442C) and in vitro (KeratinoSens, eq. to OECD TG 442D) skin sensitization assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitization. There is also by expert judgement an absence of reliable skin sensitization alerts in relevant in silico models (OASIS TIMES v2.2816). Within the DPRA assay the depletion of the CYS was determined to be due to oxidation/dimerisation by LC-MS testing with no CYS-test item adduct formation. However, conversely there is a positive result in a validated in vitro (U-SENS, OECD TG 442E) skin sensitisation assay covering Key Event 3 and/or an available LLNA in vivo assay (OECD TG 429) with EC3 28.8%. The LLNA was considered to have significant methodological issues, insofar: no evidence of the choice of dose range employed, preliminary screening and no indication as to ear thickness measurements, despite clear indications of moderate ear swelling in the first four days of test item application. It is additionally noted under OECD TG 442E paragraph 4, that for some surfactants the U-SENS positive results should be considered with caution due to high false-positive results (3 of 7) seen in the supporting literature. There are unique properties of the substance which may yield trace levels of linear unsaturated-carboxylate equivalents of the macrocyclic lactone with surfactant properties. These type of constituents (non-ionic surfactants) may have confounding issues for U-SENS non-specific CD86 expression (see: Piroird et al.,(2015): Toxicology in vitro, 29: 901-916) and/or have been demonstrated to due to non-specific proliferation confound the local lymph node assay (see: Ball et al. (2011), Regulatory Toxicology and Pharmacology, 60: 389-400 and Basketter et al. (2012), Regulatory Toxicology and Pharmacology, 64: 9-16). Potential for false positive cannot be excluded with either assay.

 

Based on the weight of evidence the applicant is applying a precautionary classification of skin sensitisation in accordance with the Regulation (EC) 1272/2008: category 1B criteria.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B.

 

The weight of evidence indicates (and application on precautionary basis) that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.

 

Weight of Evidence Conclusion:

The applicant assesses by expert judgement that GHS skin sensitisation category 1B will be applied on a precautionary basis. The weight of evidence that there is no evidence of skin sensitisation in two validated in chemico (DPRA, eq. to OECD TG 442C) and in vitro (KeratinoSens, eq. to OECD TG 442D) skin sensitization assays covering Key Event 1 and Key Event 2 in the OECD IATA AOP for skin sensitization. There is also by expert judgement an absence of reliable skin sensitization alerts in relevant in silico models (OASIS TIMES v2.2816). Within the DPRA assay the depletion of the CYS was determined to be due to oxidation/dimerisation by LC-MS testing with no CYS-test item adduct formation. However, conversely there is a positive result in a validated in vitro (U-SENS, OECD TG 442E) skin sensitisation assay covering Key Event 3 and/or an available LLNA in vivo assay (OECD TG 429) with EC3 28.8%. The LLNA was considered to have significant methodological issues, insofar: no evidence of the choice of dose range employed, preliminary screening and no indication as to ear thickness measurements, despite clear indications of moderate ear swelling in the first four days of test item application. It is additionally noted under OECD TG 442E paragraph 4, that for some surfactants the U-SENS positive results should be considered with caution due to high false-positive results (3 of 7) seen in the supporting literature. There are unique properties of the substance which may yield trace levels of linear unsaturated-carboxylate equivalents of the macrocyclic lactone with surfactant properties. These type of constituents (non-ionic surfactants) may have confounding issues for U-SENS non-specific CD86 expression (see: Piroird et al.,(2015): Toxicology in vitro, 29: 901-916) and/or have been demonstrated to due to non-specific proliferation confound the local lymph node assay (see: Ball et al. (2011), Regulatory Toxicology and Pharmacology, 60: 389-400 and Basketter et al. (2012), Regulatory Toxicology and Pharmacology, 64: 9-16). Potential for false positive cannot be excluded with either assay.

 

Based on the weight of evidence the applicant is applying a precautionary classification of skin sensitisation in accordance with the Regulation (EC) 1272/2008: category 1B criteria.