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Description of key information

Skin sensitisation (in chemico): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. The test item showed a mean depletion of lysine and cysteine peptides of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Skin sensitisation (in vitro): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test item isoborneol may be classified as skin sensitizer using the KeratinoSensTM test method.

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:
10 April 2017 - 13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Details on study design:

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, LLC; Ref. Ac RFAACAA-COOH; batch no 160801; purity 94.82%)
Lysine peptide (supplier RS Synthesis, LLC; Ref. Ac-RFAAKAA-COOH; batch no 160801; purity 94.19%)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5 ± 0.05) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2 ± 0.05) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy.
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.
Reference control C: prepared with acetonitrile, the test item and the positive control solvent, in order to check its influence on the peptide stability.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 47.2 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: 38.2 µl of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution with acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 11 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 22 ml of phosphate buffer (100 mM; pH 7.5 ± 0.05).
Lysine solution: 14.8 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 28,6 ml of ammonium acetate buffer (100 mM; pH 10.2 ± 0.05).

TEST SOLUTIONS
Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs).
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
All the replicates were prepared with the same peptide stock solutions.

1 ml of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM Sample): 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 (0.5 mM Peptide, 25 mM Sample): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

The vials were capped and mixed carefully avoiding bubbling, then placed in the HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.
Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples were checked. No precipitate was observed.

Replicates: Each sample was tested 3 times from 3 independent solutions

HPLC ANALYSIS
-Apparatus: Waters e2695 HPLC; Waters 2489 UV detector; Cortecs column C18 2.7 µm ; dimensions 2.1 x 100 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.
-Equilibration of the column: at 30°C with 50% phase A (0.1% (v/v) trifluoroacetic acid in water) and 50% phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for at least 20 minutes before running.
-Volume injected: 7 µl of each sample
-Flow rate: 0.21 ml/min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Duration of re-equilibration to the initial conditions between injections: at least 4 min.
-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 reference controls C were placed at the beginning of each repetition.
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 and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.

DEVIATIONS FROM OECD GUIDELINE: No.
The column used has 2.7 µm particle size when the column described in the OECD 442C has 3.5 µm particle size. According to the guideline, the set-up parameters were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides. Proficiency substances recommended in the OECD guideline were performed in these conditions.
Positive control results:
The depletion mean rate was 73.86% for cysteine peptide and 55.56% for lysine peptide.
Key result
Parameter:
other: %depletion in lysine peptide
Run / experiment:
mean of 3 repetitions
Value:
2.15
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: %depletion in cysteine peptide
Run / experiment:
Mean of 3 repetitions
Value:
1.62
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: mean %depletion in peptides
Run / experiment:
Mean of lysine and cysteine peptides
Value:
1.89
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: 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 0.511 mM for lysine and 0.502 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 0.86% which is lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.516 mM for lysine and 0.496 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls C was 1.16% which is lower than 15%.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 0.243% for cysteine and 0.43% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 73.86% for cysteine peptide and 55.56% 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 1.58% for cysteine and 0.82% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Table 3: Positive control

Cinnamaldehyde

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

Repetition 1

56.06

73.71

Repetition 2

55.31

73.73

Repetition 3

55.32

74.14

SD

(Standard Deviation)

0.430

0.243

Mean

55.56

73.86

Depletion

Validity criteria

40.2 < Depletion < 69.4

60.8 < Depletion < 100

CV

0.77%

0.33%

Table 4: Test item

 

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

 

Repetition 1

2.33

0

 

Repetition 2

1.26

1.71

Mean Depletion %

Repetition 3

2.86

3.16

Mean

2.15

1.62

1.89

SD

(Standard Deviation)

0.82

1.58

 

SD Validity criteria

< 11.6%

< 14.9%

 

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:
The test item shows mean depletion of 2.15% for lysine and 1.62% for cysteine, i.e. an overall average of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for isoborneol 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 as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. 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 3 times from 3 independent solutions. All validity criteria were fulfilled. The test item shows mean depletion of 2.15% for lysine and 1.62% for cysteine, i.e. an overall average of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 April 2017 - 28 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
Dilution strategy (see justification on "details on study design")
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Details on study design:

REAGENTS AND MEDIA
-DMSO (Supplier ref. D5879-1L, Sigma Aldrich; Batch no SZBG2170; purity ≥99.5%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 1852416)
-Non-heat inactivated foetal calf serum (Supplier ref. 10270098, Fisher Bioblock; Batch no 42Q9761K)
-Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin.
-Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum.
-Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum.
-Staining solution: 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS (phosphate buffered saline). Prepared extemporaneously and used within the day.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in maintenance medium.
-Passage number: cells were used at passage 25 in repetition 1 and passage 16 in repetition 2.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
-Negative (solvent) control: DMEM 1 g/l glucose, 1% DMSO, 1% non-heat inactivated foetal calf serum.

CELLS SEEDING.
-Culture plates: 5; 3 white cell culture plates (96 wells) for luminescence reading (induction measurement) + 2 transparent cell culture plates (96 wells) for absorbance reading (cytotoxicity).
-Cells suspension: 125 µl at 8x10e4 cells/ml in seeding medium were distributed in the culture plates.
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated 24 hours ± 1 hour at 37ºC, 5% CO2

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Test item solution: 2000 µM (1X) in treatment medium 1% DMSO
Positive control stock solution: The positive control was prepared at 200 mM in DMSO then diluted to the final concentration of 6.4 mM.

1) 100 X plate (positive and negative control): A 100-fold concentrated dilutions series was prepared in 96-well plate:
-Positive control: 100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl of the column 11 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Negative control: 100 µl of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12 (in cytotoxicity repetition 2, negative control was distributed in row H).

2) 4X dilution plate (positive and negative control): The 100 X plate was diluted 25 fold in a new plate (4 X).

3) Preparation of the 1X dilution for the test item:
The test item was placed in the row B.
1100 µl of treatment medium, 1% DMSO were distributed columns 1 to 10 in a masterblock. 2200 µl of the 2000 µM solution were placed in column 12 then the series dilutions were prepared by transferring 1100 µl of the column 12 in the column 11 and so until the column 1. Dilutions were mixed by repeated pipetting at least 3 times, between each concentration.

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
-Negative and positive control: In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC, 5% CO2).
-Test item: In the 5 seeded plates, the medium was aspirated. The 1X masterblock was replicated 5 times: 200 µl from the 1X masterblock were placed in each of the three white plates and in the two transparent plates. The plates (1X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC ± 1°C, 5% CO2).

REPLICATES: The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

LUCIFERASE ACTIVITY.
-Apparatus: Luminometer: GloMax™ (Promega)
-Validity of luminometer: validated according to the procedure described in Annex 3 of the OECD 442D guideline.
-Luciferase substrate: luciferine + ATP + lysing agent. Bright Glo™ Luciferase Assay System (Promega).
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

CITOTOXICITY ASSESSMENT.
-Apparatus: MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance.
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37ºC, 5% CO2). After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS (sodium dodecyl sulfate) a weekend in the dark (37°C, 5% CO2)(repetition 1) and one night in the dark (37ºC, 5% CO2) (repetition 2). After a 10 minutes homogenization, the absorbance was measured at 540 nm.

DEVIATIONS FROM OECD GUIDELINE:
The dilution strategy is different from the OECD 442D TG (paragraph 22). Given the slight solubility of the test item in water (i.e. 828 mg/l / 5.36 mM), the dilution was prepared directly 1X in treatment medium-1% DMSO at 2000 µM. This has no impact on the study result because the test item was tested in the expected final condition (i.e. 2000 µM as maximal concentration).
Positive control results:
Repetition 1: The maximal average induction of luciferase activity (Imax) was 7.90 at a concentration of 64 µM. The mean value EC1.5 was 8.24 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 2.98 at a concentration of 64 µM. The mean value EC1.5 was 19.12 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Repetition 1
Value:
2.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
Repetition 2
Value:
2.14
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5 (µM)
Run / experiment:
Repetition 1
Value:
350.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(EC1.5 < IC30)
Key result
Parameter:
other: EC1.5 (µM)
Run / experiment:
Repetition 2
Value:
394.22
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(EC1.5 < IC30)
Key result
Parameter:
other: IC30 (µM)
Run / experiment:
Repetition 1
Value:
> 2 000
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
(EC1.5 < IC30)
Key result
Parameter:
other: IC30 (µM)
Run / experiment:
Repetition 2
Value:
1 055.49
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive 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 13.6% and for repetition 2 was 9.0% which are less than 20%.
- Acceptance criteria met for positive control: Yes, the luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The average induction values at 64 µM were 7.90 and 2.98 in each repetition which are between 2 and 8. The EC1.5 values were 8.24 and 19.12 µM for each repetition which are within 2 SD of the historical mean of the testing facility and between 7 µM and 30 µM based on the OECD validation dataset.

Table 1: Positive control

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.37

1.48

2.22

3.66

7.90

8.24

7.90

Rep 2

1.15

1.19

1.22

2.67

2.98

19.12

2.98

Mean

1.26

1.33

1.72

3.17

5.44

12.55*

5.44

*geometric mean

Table 2: Test item

VIABILITY

INDUCTION

IC30(µM)

IC50(µM)

Imax

Linear EC1.5 (µM)

EC1.5 Lin/Log (µM)

Rep 1

>2000

>2000

2.17

350.57

330.40

Rep 2

1055.49

>2000

2.14

394.22

372.91

Mean

 -

 -

2,15

 -

 -

Geometric mean

 -

-

 -

371.75

351.01

Table 3: Test item mean viability percentage

Concentration µM

0,98

1,95

3,91

7,81

15,6

31,3

63

125

250

500

1000

2000

Rep 1

81,25

100,27

93,17

87,36

95,38

85,53

87,74

90,57

81,94

80,18

77,74

70,56

Rep 2

103,80

97,42

99,50

97,34

88,90

93,04

96,63

87,47

86,51

76,16

70,74

57,43

Viability

92,52

98,85

96,33

92,35

92,14

89,29

92,19

89,02

84,23

78,17

74,24

64,00

Table 4: Test item mean induction

Concentration µM

0,98

1,95

3,91

7,81

15,63

31,25

62,50

125

250

500

1000

2000

Rep 1

1,17

1,08

1,19

1,15

1,12

1,27

1,25

1,30

1,35

1,73

2,17

1,99

Rep 2

1,00

1,24

1,12

1,29

1,17

1,19

1,19

1,42

1,37

1,60

2,14

1,76

Induction

1,08

1,16

1,16

1,22

1,14

1,23

1,22

1,36

1,36

1,66

2,15

1,88

SD

0,12

0,11

0,05

0,09

0,04

0,06

0,04

0,09

0,01

0,09

0,02

0,16

Table 5: Student t-test

Rep 1

0,554

0,458

0,011

0,085

0,380

0,031

0,029

0,032

0,006

0,002

0,000

0,055

Rep 2

0,987

0,137

0,038

0,099

0,012

0,078

0,039

0,009

0,003

0,001

0,001

0,004

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 item isoborneol may be classified as skin sensitizer using the KeratinoSensTM test method.
Executive summary:

The KeratinoSensTM test method was performed for isoborneol 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 ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. For the test item, calculated Imax values were higher than 1.5, EC1.5 values were lower than 1000 µM, at EC1.5 concentration the reduction of viability was determined lower than 30% (i.e. EC1.5 < IC30) and it was found an apparent overall dose-response for luciferase induction in each repetition. Thus a positive result can be predicted for skin sensitization using the KeratinoSensTM test method.

Endpoint conclusion
Additional information:

Skin sensitisation (in chemico): Weight of evidence. A DPRA skin sensitization test was performed for isoborneol 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 as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. 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 3 times from 3 independent solutions. All validity criteria were fulfilled. The test item shows mean depletion of 2.15% for lysine and 1.62% for cysteine, i.e. an overall average of 1.89%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Skin sensitisation (in vitro): Weight of evidence. The KeratinoSensTM test method was performed for isoborneol 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 ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. For the test item, calculated Imax values were higher than 1.5, EC1.5 values were lower than 1000 µM, at EC1.5 concentration the reduction of viability was determined lower than 30% (i.e. EC1.5 < IC30) and it was found an apparent overall dose-response for luciferase induction in each repetition. Thus a positive result can be predicted for skin sensitization using the KeratinoSensTM test method.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification