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Description of key information

Skin sensitisation was investigated using GLP in vitro studies performed according to OECD TG 442C-E. The following results have been obtained:

  • OECD 442C: negative
  • OECD 442D: technically impossible due to limited solubility
  • OECD 442E: technically impossible due to limited solubility

The in vitro data are insufficient to conclude on the endpoint of skin sensitisation. Therefore, an additional in vivo LLNA study according to OECD 429 was performed. The test item was found to be a skin sensitiser and an EC3 value of 6.4% (w/w) was derived.

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:
June 02 - Nov 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Freie Handelsstadt Hamburg
Type of study:
direct peptide binding assay
Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 68.60%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.

If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Peptide Depletion in %


1) Cystein Depletion

Sample
Peak Area
Actual Peptide [mM]
Peak Are Ref Control C
Peptide Depletion [%]
Corr. peptide Depletion [%]
1
50.9758
0.503
50.8281
-0.03
0.00
2
51.5350
0.509
51.0861
-1.13
0.00
3
51.5140
0.508
50.9661
-1.09
0.00
Mean
51.342
0.507
50.9601
-0.75
0.00
Evaluation
Reactivity Class. Negative


1) Lysine Depletion

Sample
Peak Area
Actual Peptide [mM]
Peak Are Ref Control C
Peptide Depletion [%]
Corr. peptide Depletion [%]
1
36.3497
0.464
35.0532
-2.95
0.00
2
36.4728
0.465
35.3563
-3.29
0.00
3
36.5649
0.466
35.5197
-3.55
0.00
Mean
36.462 0.465
35.3097
-3.26
0.00
Evaluation
Reactivity Class. Negative


Interpretation of results:
other: The result has to be considered in an integrated approach.
Conclusions:
The test material revealed a mean cysteine and lysine peptide depletion of 0.0% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).
Executive summary:

Pupose

The purpose of this study was to determine the sensitising potential of the test material in a Direct Peptide Reactivity Assay (DPRA).

Methods

The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following incubation with the test item. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and nonsensitisers.

Results

The test item was dissolved at a concentration of 100 mM in acetone. No precipitate in the reaction mixture at the end of the incubation time and no co-elution were observed.

The test material-treated samples revealed a cysteine peptide depletion of 0.0% and a lysine peptide depletion of 0.0% (mean peptide depletion of 0.0%) and, hence, were well

below 6.38%. The test material is considered negative and predicted to be a nonsensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetone. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of

68.65% for cysteine and 59.73% for lysine peptide. These values are within the required range. Therefore, the study can be regarded as valid.

Conclusion

The test material revealed a mean cysteine and lysine peptide depletion of 0.0% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or

minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Endpoint:
skin sensitisation: in vitro
Study period:
Jun 03 - Sep 17, 2019
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The test item appeared to be insoluble in the required concentration in water. The test item was insoluble in DMSO at the required stock solution concentration of 200 mM and formed no stable suspension after addition of treatment medium. Since the KeratinoSens assay is not suitable for test items which precipitate into non stable suspensions or emulsions (defined by OECD 442D) the test item has to be considered as being technically impossible in this test.
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Freie Handelsstadt Hamburg
Type of study:
activation of keratinocytes
Details on study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Interpretation of results:
study cannot be used for classification
Conclusions:
Since the KeratinoSens assay is not suitable for test items which precipitate into non stable suspensions or emulsions (defined by OECD 442D) the test item has to be considered as being technically impossible in this test.
Endpoint:
skin sensitisation: in vitro
Study period:
Jul 29 - Aug 27, 2019
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The test item appeared to be insoluble in the required concentration in medium and DMSO. Since the h-CLAT assay is not suitable for non-stable suspensions or emulsions the test system has to be considered unsuitable for this test item.
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Gesundheit und Verbraucherschutz, Freie Handelsstadt Hamburg
Type of study:
activation of dendritic cells
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item appeared to be insoluble in the required concentration in medium and DMSO. Since the h-CLAT assay is not suitable for non-stable suspensions or emulsions the test system has to be considered unsuitable for this test item.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Dec 04, 2019 - Feb 18, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test test: 8 - 9weeks
Main Test: 9 - 10 weeks
- Weight : 17.0 - 23.6 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 20-70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6
Vehicle:
propylene glycol
Concentration:
5, 10, and 25 (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 50% in MEK

Range Finding Exp. 1
- Concentration used: 25, 50 %
- Irritation:erythema of the ear skin (Score up to 3)

Range Finding Exp. 2
- Concentration used: 5, 10 %
- Irritation:very slight erythema of the ear skin (Score 1)



MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Concentration: 2.5, 5, and 10%
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Standard statistical methods have been applied for data processing.
Positive control results:
Conc. SI
0%: 1.0
5% 1.68
10% 1.78
25% 8.19
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
Test Group: 2.5% in MEK
Key result
Parameter:
SI
Value:
2.5
Test group / Remarks:
Test Group: 5% in MEK
Key result
Parameter:
SI
Value:
4.3
Test group / Remarks:
Test Group: 10% in MEK
Key result
Parameter:
EC3
Value:
6.4
Cellular proliferation data / Observations:

EC3 CALCULATION : EC3 = (a-c) [(3-d)/(b-d)] + c; a,b,c,d = coordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot

CLINICAL OBSERVATIONS: The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with test item concentrations of 5 and 10% showed a very slight erythema of the ear skin (Score 1). Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation of Stimulation Indices per Dose Group

Test item concentration
Group Calculation
Mean DPM per animal (2 lymph nodes)
SD
S.I.
MEK (Vehicle Control)
1415.6
231.6
1.0
2.5 % Test Item in MEK
2166.8
714.2
1.5
5 % Test Item in MEK 3574.4
764.0
2.5
10 % Test Item in MEK 6064.6
2110.7
4.3


Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was found to be a skin sensitiser and an EC3 value of 6.4% (w/w) was derived.
Executive summary:

Objective

In the study the test item formulated in methyl ethyl ketone (MEK) was assessed for its possible skin sensitising potential.

Study Design

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10 (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

Results

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

The animals treated with test item concentrations of 5 and 10% showed a very slight erythema of the ear skin (Score 1). Animals treated with 2.5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 1.5, 2.5, and 4.3 were determined with the test item at concentrations of 2.5, 5, and 10% in MEK, respectively. A clear dose response was observed.

Conclusion

The test item was found to be a skin sensitiser and an EC3 value of 6.4% (w/w) was derived.

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

In vitro skin sensitisation

The KeratinoSens Assay as well as the h-Clat are technical impossible due to the limited solubility of the test item in the solvents according to the appropriate OECD Guideline.

In the Directive Peptide Reactivity Assay (DPRA), the test material revealed a mean cysteine and lysine peptide depletion of 0.0% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) .

However, the in vitro data are insufficient for a conclusion on classification an labelling and therefore an in vivo LLNA assay according to OECD 429 was conducted.

In vivo skin sensitization test

In this study Stimulation Indices (S.I.) of 1.5, 2.5, and 4.3 were determined with the test item at concentrations of 2.5, 5, and 10% in MEK, respectively. A clear dose response was observed. The test item was found to be a skin sensitiser and an EC3 value of 6.4% (w/w) was derived.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information a classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures for skin sensitisation in Class 1B (H317) is justified.