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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (in chemico): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. Under the experimental conditions, the test substance was concluded as sensitizer in DPRA assay.

Skin sensitisation (in vitro): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test substance may be classified as not skin sensitizer using the KeratinoSensTM test method.

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the“2 out of 3-sens ITS” approach as described in annex 1 of OECD guidance ENV/JM/MONO(2016)29, two concordant results addressing two different key events (KEs) indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser. Since no two concordant results have been obtained from the tests performed, another in vitro skin sensitisation test must be conducted in order to allow the final classification of the substance. However, as a conservative estimate, the test substance is considered a sensitizer cat. 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
maximization test
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
A maximization test (Kligman, 1966; Kligman & Epstein, 1975) was carried out on 25 volunteers.
No more data was provided on the method.
GLP compliance:
no
Type of study:
other: maximization test
Species:
other: human
Strain:
other: Not applicable
Sex:
not specified
Reading:
1st reading
Group:
test chemical
Dose level:
10 % in petrolatum
Remarks on result:
no indication of skin sensitisation

The material was tested at a concentration of 10 % in petrolatum and produced no sensitization reactions.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The material was tested on humans at a concentration of 10% in petrolatum and produced no sensitization reactions.
Executive summary:

A maximization test was carried out on 25 volunteers. The material was tested at a concentration of 10% in petrolatum and produced no sensitization reactions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
maximization test
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
A maximization test (Kligman, 1966; Kligman & Epstein, 1975) was carried out on 25 volunteers.
No more data was provided on the method.
GLP compliance:
no
Type of study:
other: maximization test
Species:
other: human
Strain:
other: Not applicable
Sex:
not specified
Reading:
1st reading
Group:
test chemical
Dose level:
4 % in petrolatum
Remarks on result:
no indication of skin sensitisation

The material was tested at a concentration of 4 % in petrolatum and produced no sensitization reactions.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The material was tested on humans at a concentration of 4 % in petrolatum and produced no sensitization reactions.
Executive summary:

A maximization test was carried out on 25 volunteers. The material was tested at a concentration of 4 % in petrolatum and produced no sensitization reactions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils.
No more data provided on the method.
GLP compliance:
no
Type of study:
patch test
Species:
other: human
Strain:
other: Not applicable
Sex:
not specified
Route:
other: No data
Vehicle:
other: No data
Concentration / amount:
no data
Route:
other: No data
Vehicle:
other: No data
Concentration / amount:
No data
No. of animals per dose:
No data
Positive control substance(s):
not specified
Reading:
1st reading
Group:
test chemical
Remarks on result:
no indication of skin sensitisation

alpha pinene was found not to be a sensitizer for human skin.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Alpha pinene was found not to be a sensitizer for human skin.
Executive summary:

A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils. Alpha pinene was found not to be a sensitizer for human skin.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils.
No more data provided on the method.
GLP compliance:
no
Type of study:
patch test
Species:
other: human
Strain:
other: Not applicable
Sex:
not specified
Route:
other: No data
Vehicle:
other: No data
Concentration / amount:
no data
Route:
other: No data
Vehicle:
other: No data
Concentration / amount:
No data
No. of animals per dose:
No data
Positive control substance(s):
not specified
Reading:
1st reading
Group:
test chemical
Remarks on result:
no indication of skin sensitisation

Camphene was found not to be a sensitizer for human skin.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Camphene was found not to be a sensitizer for human skin.
Executive summary:

A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils. Camphene was found not to be a sensitizer for human skin.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
maximization test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha pinene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Reading:
1st reading
Group:
test chemical
Dose level:
10 % in petrolatum
Remarks on result:
no indication of skin sensitisation

Based on read-across approach form analogue alpha pinene tested at a concentration of 10 % in petrolatum, the reaction mass is predicted to not produce sensitization reactions.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on read-across approach form analogue alpha pinene tested on humans at a concentration of 10 % in petrolatum, the reaction mass is predicted to not produce sensitization reactions.
Executive summary:

A maximization test was carried out on 25 volunteers. The material was tested at a concentration of 10% in petrolatum and produced no sensitization reactions. Based on these results, the read-across approach was applied and the reaction mass is predicted to not produce sensitization reactions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
maximization test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Reading:
1st reading
Group:
test chemical
Dose level:
4 % in petrolatum
Remarks on result:
no indication of skin sensitisation

Based on read-across approach form analogue camphene tested at a concentration of 4 % in petrolatum, the reaction mass is predicted to not produce sensitization reactions.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on read-across approach form analogue camphene tested on humans at a concentration of 4 % in petrolatum, the reaction mass is predicted to not produce sensitization reactions.
Executive summary:

A maximization test was carried out on 25 volunteers. The material was tested at a concentration of 4 % in petrolatum and produced no sensitization reactions. Based on these results, the read-across approach was applied and the reaction mass is predicted to not produce sensitization reactions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha pinene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Reading:
1st reading
Group:
test chemical
Remarks on result:
no indication of skin sensitisation

Based on read-across approach form analogue alpha pinene, the reaction mass is not expected to be a sensitizer for human skin.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on read-across approach from analogue alpha pinene, the reaction mass is not expected to be a sensitizer for human skin.
Executive summary:

A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils. Alpha pinene was found not to be a sensitizer for human skin. Based on these results, the read-across approach was applied and the reaction mass is not expected to be a sensitizer for human skin.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Reading:
1st reading
Group:
test chemical
Remarks on result:
no indication of skin sensitisation

Based on read-across approach form analogue camphene, the reaction mass is not expected to be a sensitizer for human skin.

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on read-across approach form analogue camphene, the reaction mass is not expected to be a sensitizer for human skin.
Executive summary:

A Patch-Test was performed on humans for stuying the sensitizing properties of 17 terpenes and related compounds present in essential oils. Camphene was found not to be a sensitizer for human skin. Based on these results, the read-across approach was applied and the reaction mass is not expected to be a sensitizer for human skin.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 January 2018 - 11 February 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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
Details on study design:

REAGENTS AND MEDIA
-Fetal Bovine Serum (FBS): Gibco (Lot # 1789459)
-DMSO: Qualigens (Lot # 2217110817)
-D-MEM with Glutamax: Gibco (Lot # 1897167)
-Ethylene Diamine Tetra Acetic Acid (EDTA): Sigma (Lot # SLBM9213V)
-DPBS (1X): Gibco (Lot # RNBF8606)
-Trypsin-EDTA: Gibco (Lot #1894157)
-Geneticin: Gibco(Lot #1751936)
-Culture medium: D-MEM with Glutamax supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin.
-Exposure medium: D-MEM supplemented with 1% foetal bovine serum (495 mL D-MEM + 5 mL FBS).
-Staining solution: MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in culture medium.
-Passage number: cells were used at passage 21 (experiment 1 and 2) and passage 24 (experiment 3).

CONTROLS
-Positive control: cinnamaldehyde (CAS 14371-10-9; batch no STBF4119V; purity 99.4%)
-Negative (solvent) control: DMSO

CELLS SEEDING.
-Cells suspension: Cells were maintained in culture medium. After cells attained 80-90% confluency, they were washed twice with DPBS containing 0.05% of EDTA. To each flask 2-3 mL of 0.05% Trypsin-EDTA was added and flasks were incubated at 37 ± 1ºC (usually 5–10 minutes). After cells were detached completely, they were resuspended in DMEM with 9.1% FBS without geneticin. Cell count was taken and cell density was adjusted to 8 x 10e4 cells/mL. 125 µL of this culture (containing approx. 10,000 cells) was then dispensed in each well of 96 well plates, leaving one cell empty (to assess background values).
-nº of culture plates: four; three 96-well white assay plates (for luciferase assay) and one 96-well flat bottom transparent plate (for cytotoxicity, MTT assay).
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated for 24 hours in 5±1 % CO2 at 37±1ºC.

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Positive control: Trans cinnamaldehyde was dissolved in DMSO to achieve concentration of 200 mM. This solution was further diluted to final concentration of 6.4 mM by adding 32 µL of 200 mM solution to 968 µL of DMSO.
Negative control: DMSO was used as negative (solvent) control.
Test Item Preparation: A quantity of 40 mg of test item was dissolved in 1 mL DMSO to attain the stock solution concentration of 40 mg/mL.
Preparation of 100X Master Plate: A 100-fold concentrated dilutions series was prepared in 96-well plate:
-For Test Item Row (Row A): 100 µL of DMSO was added in column 1-11 of rows A. Then 100 µL of 40 mg/mL of stock solution of test item was added in column 11 & 12 of corresponding rows. Serial dilutions were prepared by transferring 100 µL from column 11 to column 10 and was continued till column 1.
-For Control Row (Row H): 100 µL of DMSO was added in column 1- 10 and column 12. In column 10 and 11, 100 µL of 6.4 mM stock solution of trans cinnamaldehyde was added. Serial dilutions were prepared by transferring 100 µL from column 10 to column 9 (till column 7). Column 1-6 served as negative control (DMSO), column 7-11 served as positive control and column 12 served as no cell blank

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
After 24 ± 2 hours of incubation, medium from all the 4 plates (for single test item) was aspirated and discarded. It was replaced with 150 µL of DMEM containing 1% FBS without geneticin. The 25 fold dilution of the 100X master plate was performed into a fresh plate (10 µL test solution + 240 µL of DMEM with 1% FBS without geneticin). Resulting 4X plate was further distributed to assay plates: 50 µL of these stock solutions were added 3 white assay plates and one cytotoxicity plate already containing 150 µL of culture medium. Plates were then covered with foil or petri-seal and incubated for 46 hours(expt-1 and 2) and 47 hours(expt-3) in 5 ± 1 % CO2 at 37 ± 1ºC.

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.1953125 µg/mL to 400 µg/mL.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.


REPLICATES: The study was composed of 3 independent repetitions. For each repetition the test item and the controls were replicated on three independent plates for the measurement of induction and one plate for the measurement of cytotoxicity. Two valid repetitions (experiment 1 and 3) were considered for final evaluation The reason for conducting 3 experiments was the failure of positive controls to meet the acceptance criteria (experiment 2).

LUCIFERASE ACTIVITY.
-Procedure: After 48 ± 2 hours of incubation, the medium from 96 well white assay plates was aspirated and discarded. Cells were washed once with 150 µL of DPBS. After washing, 20 µL of passive lysis buffer was added in each well. Cells were then incubated for 30 ± 10 minutes at 37 °C. After incubation, the plates with cell lysate were placed in luminometer. 50 µL of 1X Luciferase substrate was added and luciferase activity was recorded for 2 seconds.

CITOTOXICITY ASSESSMENT.
-Procedure: Master mix was prepared by combining 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin. After 48 ± 2 hours of incubation, the medium from 96 well transparent plate was replaced with 227 µL above prepared master mix. The plate was covered with foil and incubated for 4 hours. After incubation, the medium was removed and 200 μL of a 10% SDS solution was added to each well. The plate was sealed and covered with a foil and placed in the incubator for overnight incubation. Following incubation the absorption at 570 nm was measured.

DEVIATIONS FROM OECD GUIDELINE: No.
Positive control results:
The gene induction for positive control was found to be >1.5 at concentrations of 32 µM and 64 µM in both the repetitions. The EC1.5 value for positive control was found to be 36.37 µM and 26.21 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 1.88 and 7.30 for experiment 1 and 3, respectively. Clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 3.
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: Imax
Value:
0.98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Repetition 3
Parameter:
other: Imax
Value:
1.16
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: EC1.5 (µg/mL)
Value:
200
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
(EC1.5 > IC30)
Key result
Run / experiment:
other: Repetition 3
Parameter:
other: EC1.5 (µg/mL)
Value:
200
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
(EC1.5 > IC30)
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: IC30 (µg/mL)
Value:
20.03
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
(EC1.5 > IC30)
Key result
Run / experiment:
other: Repetition 3
Parameter:
other: IC30 (µg/mL)
Value:
20.02
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
(EC1.5 > IC30)
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 9.66% and for repetition 3 was 14.74% which are less than 20%.
Variability in the experiment 3 was in fact 22.25 %. Grubbs' test was applied and since the deviation was higher due to a single outlier in the 18 wells, results are considered valid. After removal of outlier variation in the negative control was 14.74%

- Acceptance criteria met for positive control: The luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The EC1.5 value for positive control was found to be 36.37 µM and 26.21 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 1.88 and 7.30 for experiment 1 and 3, respectively. The latter criterion is not fulfilled, however, clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 3. Thus, the acceptance criteria are met.



Table 1: Mean Summary of Imax, EC1.5, IC30 and IC50 values of Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)  

Imax

Test Item

Rep1

Rep2

Average

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)

0.98

1.16

1.07

 

EC1.5

Test Item

Rep1

Rep2

GeoMean

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)

>200µg/mL

>200µg/mL

>200µg/mL

 

IC50

Test Item

Rep1

Rep2

GeoMean

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)

20.05µg/mL

20.06µg/mL

20.05µg/mL

 

IC30

Test Item

Rep1

Rep2

GeoMean

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)

20.03µg/mL

20.02µg/mL

20.03µg/mL

Table 2: Mean Summary of Imax, EC1.5, IC30 and IC50 values of Positive control

T-Cinnamaldehyde

Imax

EC1.5

IC30

IC50

Rep 1

1.88

36.37

-

-

Rep 2

7.30

26.21

-

-

Table 3: Summary of Mean Induction of Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene) (Two Experiments)

Concentration

(µg/mL)

Reaction Mass (Fenchene, Laevo Alpha Pinene, Laevo Camphene, Dextro Camphene)

Mean

SD

0.195313

1.06

0.14

0.390625

0.94

0.22

0.78125

0.88

0.08

1.5625

0.96

0.03

30125

1.05

0.15

6.25

0.95

0.00

12.5

0.86

0.04

25

0.11

0.13

50

-0.01

0.00

100

0.01

0.00

200

0.02

0.00

400

0.03

0.01
Interpretation of results:
other: KeratinoSensTM test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.
Conclusions:
Under the experimental conditions the test substance may be classified as not skin sensitizer using the KeratinoSensTM test method.
Executive summary:

The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.195 µg/mL to 400 µg/mL, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 3 independent repetitions and out of these, two valid repetitions (experiment 1 and 3) were considered for final evaluation. The reason for conducting 3 experiments was the failure of positive controls to meet the acceptance criteria (experiment 2). All validity criteria were fulfilled. For the test item, calculated Imax values were lower than 1.5 and EC1.5 values were higher than 200 µg/mL in the 2 repetitions. Also at EC1.5 concentration the reduction of viability was determined higher than 30% (i.e. EC1.5 > IC30). Thus a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
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))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
TEST ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was acetonitrile.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)
Lysine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (750 µL of phosphate buffer (pH 7.5) + 200 µL of acetonitrile + 50 µL test item) for cysteine peptide and ammonium acetate buffer (750 µL of ammonium acetate buffer (pH 10.2) + 250 µL of test item) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy (750 µL Cysteine/Lysine peptide (0.667 mM) + 250 µL of acetonitrile).
Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time (same as preparation for reference control A, but run before and after 24 ± 2 hours incubation at 25 ± 2.5ºC in dark).
Reference control C: Acetonitrile was used as a vehicle hence reference control C was not used in this study. Therefore reference control B for acetonitrile was used as control C.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 40.87 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with acetonitrile.
Positive control solution: 38.10 µL of Cinnemaldehyde was dissolved in 2961.9 µL of acetonitrile for the preparation of 100 mM solution (3 mL) for both Cysteine and Lysine peptides.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 9.145 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 18.253 ml of phosphate buffer (pH 7.5).
Lysine solution: 9.084 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 17.537 ml of ammonium acetate buffer (pH 10.2).

TEST SOLUTIONS
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
1 ml of each solution was prepared according to the following quantities:
-Cysteine test solution: 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of sample or solvent for Reference controls + 200 µl of acetonitrile.
-Lysine test solution: 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

Vials were caped, vortexed and were placed in the HPLC auto sampler at 25 ± 2.5ºC in dark for 24 ± 2 hours. HPLC analysis of the batch of samples was started 24 ± 2 hours after the test item was added to the peptide solution.

Replicates: Samples were prepared in triplicate for both peptides.

HPLC ANALYSIS
-Apparatus: Shimadzu with UV Detector. Column: ZorbaX SB-C18 (2.1 mm x 100 mm x 3.5 micron) with Guard Column Phenomenex Security Guard C18 (4 mm x 2 mm)
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Volume injected: 5 µl of each sample
-Run time: 20 min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Analysis sequence:
The analysis was programmed according to the following principles:
The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.
Lysine and cysteine analysis were conducted on separate day. Samples were visually inspected for precipitation prior to HPLC analysis.

DEVIATIONS FROM OECD GUIDELINE: No.

Positive control results:
The depletion mean rate was 73% for cysteine peptide and 44% for lysine peptide.
Key result
Run / experiment:
other: mean of 3 repetitions
Parameter:
other: %depletion in lysine peptide
Value:
2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: Mean of 3 repetitions
Parameter:
other: %depletion in cysteine peptide
Value:
14
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: Mean of lysine and cysteine peptides
Parameter:
other: mean %depletion in peptides
Value:
8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.49 mM for lysine and 0.54 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 0.85 % for Cysteine peptide and 0.30% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for reference control C (control B was used instead as acetonitrile was used as solvent): yes, the mean concentration of peptide was 0.50 mM for lysine and 0.52 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls B was 0.85 % for Cysteine peptide and 0.30% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 3.20% for cysteine and 2.67% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 73% for cysteine peptide and 44% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 0.65% for cysteine and 0.86% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Table 1: Positive Control and Test Item Percent Peptide Depletion

Percent Peptide Depletion (Cysteine)

Positive Control Name/

Test Item Code

Rep-1

Area

(AU)

Rep-2

Area

(AU)

Rep-3

Area

(AU)

Average

%

Depletion

SD

RCV

Expected

Depletion

Cinnamic aldehyde

(19.05.2018)

508351

483161

479613

490375

73

15668.42

3.20

60.8-100

Test item

(19.05.2018)

1599225

1584854

1579020

1587700

14

10398.74

0.65

-

Co-elution control of
Test item

(19.05.2018)

ND

-

-

-

-

-

-

-

Percent Peptide Depletion (Lysine)

Cinnamic aldehyde

(22.05.2018)

969651

966324

923944

953306

44

25482.88

2.67

40.2-69

Test item

(22.05.2018)

1698882

1673342

1674559

1682261

2

14407.06

0.86

-

Co-elution control- Test item

(22.05.2018)

ND

-

-

-

-

-

-

-

Keys: Rep = Replicate, AU = Arbitrary Units, SD = Standard Deviation, RCV = Relative Coefficient of Variability, ND = Not Detected, - = Not applicable

No co-elution occurred of the test item neither with lysine nor with cysteine peptides.

Interpretation of results:
other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.
Conclusions:
under the experimental conditions, the test substance was concluded as sensitizer in DPRA assay.


Executive summary:

A DPRA skin sensitization test was performed on the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile. Reference controls A and B were prepared with acetonitrile (therefore control B was used as control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. The test item shows mean depletion of 2% for lysine and 14% for cysteine, i.e. an overall average of 8%, reflecting low reactivity and thus a positive prediction of DPRA.

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

Skin sensitisation (in chemico): Weight of evidence. A DPRA skin sensitization test was performed on the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile. Reference controls A and B were prepared with acetonitrile (therefore control B was used as control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. The test item shows mean depletion of 2% for lysine and 14% for cysteine, i.e. an overall average of 8%, reflecting low reactivity and thus a positive prediction of DPRA.

Skin sensitisation (in vitro): Weight of evidence. The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.195 µg/mL to 400 µg/mL, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 3 independent repetitions and out of these, two valid repetitions (experiment 1 and 3) were considered for final evaluation. The reason for conducting 3 experiments was the failure of positive controls to meet the acceptance criteria (experiment 2). All validity criteria were fulfilled. For the test item, calculated Imax values were lower than 1.5 and EC1.5 values were higher than 200 µg/mL in the 2 repetitions. Also at EC1.5 concentration the reduction of viability was determined higher than 30% (i.e. EC1.5 > IC30). Thus a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the OECD Guidance Document on the reporting of defined approaches to be used within integrated approaches to testing and assessment for skin sensitization ((ENV/JM/MONO(2016)29), the “2 out of 3-sens ITS” approach as described in its annex 1 has been selected to allow the classification of the substance.

The combination of test methods used in this strategy covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)].

As there is no differential weighting of the individual test methods used and no predefined sequential order of testing, the order and information source from which data is obtained is not defined. Due to the higher complexity and resources needed (e.g. flow cytometer needed) to conduct the tests used for KE 3, the DPRA (KE1) and Nrf2-ARE-based tests (KE2) will usually be conducted first.

Based on the current knowledge, information obtained from peptide reactivity, whether obtained from in chemico or in silico methods, seems to show the highest predictive power and may provide more weight to the overall assessment of skin sensitisation (Natsch et al., 2013, Urbisch et al., 2015).

According to this approach two concordant results addressing two different key events (KEs) indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser.

Since no two concordant results have been obtained from the tests performed, another in vitro skin sensitisation test must be conducted in order to allow the final classification of the substance. However, as a conservative estimate, the test substance is considered a sensitizer cat. 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance needs to be classified for skin sensitization according to CLP Regulation no. 1272/2008.