Juniper, Juniperus oxycedrus, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 October 2019 - 14 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See explanation in "Details on study design"

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Juniper, Juniperus oxycedrus, ext.

EC Number:

289-969-0

EC Name:

Juniper, Juniperus oxycedrus, ext.

Cas Number:

90046-02-9

IUPAC Name:

Empyreumatic oil obtained from Juniperus oxycedrus (Cupressaceae) wood by dry distillation

Test material form:

liquid

Details on test material:

- Appearance: brown liquid
- Density (20ºC): 1.017
- Refractive index (20ºC): 1.533

In vitro test system

Details on the study design:

Details on study design:

TEST SYSTEM
KeratinoSens cell line with a stable insertion of the Luciferase-construct was specially designed for this test system by Givaudan (Switzerland). The cells were supplied by Givaudan at passage 6 and cultured by the lab until the start experiment. During the experiment the cells were in passage 23 (run 1) and passage 24 (run 2).

REAGENTS AND MEDIA
- Maintenance medium: Dulbecco’s Modified Eagle Medium (DMEM) with 1000 mg/L D-Glucose and sodium pyruvate (Sigma-Aldrich, cat. no. D5546, lot no. RNBH6985), 9,1% fetal bovine serum (FBS/FCS) (Capricorn, cat. no. FBS-12A, lot no. CP18-2302), 2 mM L-Glutamine (Sigma-Aldrich, cat. no. G7513, lot no. RNBG8374) and 500 μg/mL geneticin (G-418) (Gibco, cat. no. 11811-023, lot no. 1839528).
- Exposure medium: Dulbecco’s Modified Eagle Medium (DMEM) with 1000 mg/L D-Glucose and sodium pyruvate (Sigma-Aldrich, cat. no. D5546, lot no. RNBH6985) 2 mM L- Glutamine (Sigma-Aldrich, cat. no. G7513, lot no. RNBG8374) and 1 % fetal bovine serum (FBS/FCS) (Capricorn, cat. no. FBS-12A, lot no. CP18-2302).
- DPBS (Dulbecco's Phosphate-Buffered Saline without calcium chloride and without magnesium chloride) for washing of cells was produced by Sigma-Aldrich (cat. no. D8537, lot no. RNBH2606).
- Negative (solvent) control: dimethyl sulfoxide (DMSO; CAS No. 67-68-5; IUPAC name: methylsulfinylmethane; molecular weight: 78.129 g/mol; molecular formula: C2H6OS; purity: 99.97 %; manufacturer: VWR, cat. no. 282164K, lot no. 17H044005).
- Positive control: trans-cinnamaldehyde (CAS No. 14371-10-9; IUPAC name: (E)-3-phenylprop-2- enal; molecular weight: 132.162 g/mol; molecular formula: C9H8O; purity: 99.1 %; manufacturer: Sigma- Aldrich, cat. no. 239968, lot no. STBG020V).
- Luciferase Assay System (Promega, cat no. E1501, lot no. 369690) and lysis buffer – Passive Lysis Buffer (Promega, cat. no. E1941, lot no. 0000330200)
- MTT stock solution, contained 5 mg/ml thiazolyl blue tetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, CAS No. 298-93-1; manufacturer: Sigma-Aldrich, Lot. No. MKCD8033) in DPBS.
- 10 % SDS solution (sodium dodecyl sulphate, CAS No. 151-21-3, manufacturer: VWR, cat. no. 27926.238, lot no.18A224105)

DEMOSTRATION OF PROFICIENCY
Prior to routine use, the validity of the KeratinoSens assay was demonstrated in a proficiency study. In this study, 10 proficiency chemicals (indicated by the OECD 442D guideline) were tested.
Historical data for positive control were available, which demonstrated the reliability and the validity of results.

PREPARATION OF THE TEST ITEM CONCENTRATIONS
The test item should be dissolved to a final concentration of 40 mg/ml in DMSO. Before testing, the solubility in DMSO of test item at this concentration was checked. The test item was soluble in DMSO at a concentration of 40 mg/ml. The DMSO solutions can be considered self-sterilising, so that no sterile filtration was needed in case of the test item is soluble in DMSO.
Based on the stock solutions of the test item, serial double dilutions was made using DMSO to obtain 12 master concentrations of the chemical to be tested (0.02 mg/ml to 40 mg/ml). The master concentrations were further diluted 25-fold in exposure medium, and finally used for treatment with a further 4-fold dilution factor so that the final concentrations of the test item range from (0.20 µg/ml to 400 µg/ml (0,20 µg/mL, 0,39 µg/mL, 0,78 µg/mL, 1,56 µg/mL, 3,13 µg/mL, 6,25 µg/mL, 12,5 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL).

NUMBER OF REPLICATIONS
Three replicates for each concentrations of test item were used for the luciferase activity measurements and for the cell viability assay. Two independent repetitions (runs) containing each three replicates (i.e. n=6) were performed to derive a prediction (positive or negative). The third independent run was not necessary. Each independent repetition (runs) was performed the same day with fresh stock solution of test chemicals and independently harvested cells. Cells came from the same passage.

CELL SEEDING AND EXPOSURE
The cells after thawing were cultured in sterile conditions according to internal lab method. At the time of seeding the cells were 90 % confluent (for both runs). The cells were washed twice with PBS (without Ca2+/Mg2+). Afterwards the cells were trypsinized (trypsin + EDTA) until the cells detached. To stop this reaction, maintenance medium was added. After centrifugation (5 min at 125 × g), the supernatant was discarded and the cells were resuspended in maintenance medium without geneticin. After quantification by cell counter Countess II FL, the cell suspension was adjusted to a density of 80 000 cells per mL. 125 µL of the cell suspension (10 000 cells) were seeded into each well on clear transparent flat bottom 96-well plate and on white flat bottom 96-well plate. Four wells per plate was left empty (no cells and no treatment) to assess background values – “blank”. The plates were incubated at 37 ± 1 ºC and 5.0 ± 1 % CO2 in a humidified atmosphere.
After the 24 ± 2 hours of incubation (23 hours and 50 minutes for the first run and 22 hours and 10 minutes for the second run), the medium was removed from the cells and 150 μL exposure medium was added to each well. Afterwards 50 μL of each single test item concentration and the controls (25 times diluted master concentrations) were added. All plates were then covered with a foil to avoid evaporation of volatile compounds and to avoid cross-contamination between wells by volatile compounds. Next, the treated plates were incubated at 37±1°C in the presence of 5% ± 1 % CO2.

LUCIFERASE ACTIVITY MEASUREMENTS
After 48 ± 2 hours of incubation (47 hours and 45 minutes for the first run and 47 hours and 15 minutes for the second run), the cells were washed once with DPBS, and 20 µl relevant lysis buffer for luminescence readings added to each well for 20 minutes at room temperature. Next 50 µl luciferase substrate was added to each well and the luminescence was measured on microplate reader FLUOStar Omega 5 minutes after the first addition of substrate.

CYTOTOXICITY ASSESSMENT
For the KeratinoSens™ cell viability assay, medium was replaced after the 2 days exposure (48 ± 2 hours) with fresh exposure medium containing about 0,6 mg/ml MTT and cells incubated at 37 ± 1 °C in the presence of 5 ± 1 % CO2. After 4 hours incubation, the medium was removed and 200 µl of a 10% SDS solution was added to each well. The plates were covered with a sealing tape and placed protected from light in the incubator. After at least overnight incubation (1 day for the first run and 3 days for the second runs), the absorption at 600 nm was determined for each well using microplate reader FLUOStar Omega.

DEVIATIONS FROM OECD GUIDELINE:
- An alternative luminescence measurement is used. This is related to the use of another microplate reader than described in the guideline. Nevertheless, control experiments (validation study) were carried out, which were successfully completed.
- The following MTT concentration was used: 0.6 mg / ml, instead of 5 mg / ml, which is consistent with the "DB-ALM no. 155: KeratinoSensTM ". The concentration given in the OECD guideline is too high and can adversely affect cells. As standard, a lower concentration is used in the MTT assay (eg in ISO 10993-5: 2009 (E) there is 1 mg/ml, but in twice lower volume, in OECD TG no. 491 there is 0.5 mg/ml).
Above deviations did not affect the course and the results of the study.

Results and discussion

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 7.1 at a concentration of 64 µM. The mean value EC1.5 was 5.4 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 8.7 at a concentration of 64 µM. The mean value EC1.5 was 6.5 µM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Repetition 1

Parameter:

other: Imax

Value:

3.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Repetition 2

Parameter:

other: Imax

Value:

5.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

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 and correctly categorized.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 15.5% and for repetition 2 was 9.4% which are not higher 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 EC1.5 values were 5.4 and 6.5 µM for each repetition which are within 2 SD of the historical mean of the testing facility. The average induction for the first run in the three replicates at 64 µM was between 2 and 8. However, in the second run the highest induction of luciferase was 8.7. Nevertheless, the clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control was observed in the both runs.
- Range of historical values if different from the ones specified in the test guideline: see table below.

Any other information on results incl. tables

Table 2. OD 600 (blank corrected) and viability [%] for cells treated with the test item.

conc. [μg/mL]		0.2	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
run 1	rep.1	0.613	0.552	0.664	0.704	0.810	0.865	1.068	0.065	0.028	0.080	0.031	0.037
	rep.2	0.670	0.636	0.750	0.745	0.974	1.115	0.848	0.031	0.024	0.028	0.029	0.036
	rep.3	0.683	0.745	0.715	0.764	0.925	0.979	1.001	0.034	0.030	0.023	0.030	0.034
	Mean	0.655	0.644	0.710	0.738	0.903	0.986	0.972	0.043	0.027	0.044	0.030	0.036
	SD	0.037	0.097	0.043	0.031	0.084	0.125	0.113	0.019	0.003	0.032	0.001	0.002
	blank	0.060 ± 0.034
	Viab.[%]	92.1	90.6	99.8	103.7	127.0	138.7	136.7	6.1	3.8	6.1	4.2	5.0
run 2	rep.1	0.514	0.483	0.589	0.504	0.612	0.673	0.192	0.027	0.021	0.027	0.035	0.042
	rep.2	0.529	0.386	0.552	0.645	0.678	0.533	0.073	0.021	0.021	0.024	0.029	0.035
	rep.3	0.502	0.488	0.479	0.645	0.695	0.795	0.541	0.035	0.026	0.025	0.030	0.037
	Mean	0.515	0.453	0.540	0.598	0.662	0.667	0.269	0.028	0.023	0.026	0.032	0.038
	SD	0.014	0.058	0.056	0.081	0.044	0.131	0.243	0.007	0.003	0.002	0.003	0.004
	blank	0.067 ± 0.040
	Viab.[%]	97.2	85.4	102.0	112.9	124.9	125.9	50.8	5.3	4.3	4.8	6.0	7.2

conc. = concentration, rep. = replicate (absorbance value), SD = standard deviation, Viab. = viability [%]

Table 3. OD 600 (blank corrected) and viability [%] for cells treated with positive controls.

concentration [μM]		4	8	16	32	64
run 1	rep.1	0.733	0.842	0.720	0.653	0.081
	rep.2	0.764	0.831	0.895	0.685	0.117
	rep.3	0.712	0.884	0.809	0.681	0.082
	Mean	0.736	0.852	0.808	0.673	0.093
	SD	0.026	0.028	0.088	0.017	0.021
	Viab. [%]	103.5	119.8	113.6	94.6	13.1
run 2	rep.1	0.609	0.593	0.441	0.341	0.331
	rep.2	0.581	0.616	0.463	0.552	0.140
	rep.3	0.555	0.607	0.567	0.545	0.433
	Mean	0.582	0.606	0.491	0.480	0.302
	SD	0.027	0.012	0.067	0.120	0.149
	Viab. [%]	109.8	114.3	92.6	90.5	56.9

rep. = replicate (absorbance value), SD = standard deviation, Viab. = viability [%]

Table 4. OD 600 for cells treated with negative control.

well no.		1	2	3	4	5	6	7
run 1	rep.1	0.640	0,645	0,743	0,671	0,736	0,749	0,676
	rep.2	0.597	0,641	0,709	0,738	0,729	0,718	0,716
	rep.3	0.659	0,776	0,815	0,721	0,777	0,751	0,728
	Mean	0.711
	SD	0.054
	CV [%]	7.60
run 2	rep.1	0.500	0,526	0,471	0,565	0,537	0,528	0,545
	rep.2	0.508	0,492	0,486	0,661	0,570	0,562	0,531
	rep.3	0.526	0,425	0,511	0,539	0,556	0,510	0,573
	Mean	0.530
	SD	0.047
	CV [%]	8.85

rep. = replicate (absorbance value), SD = standard deviation, Viab. = viability [%]

Table 5. IC50 and IC30 obtained for the positive control and the test item

		Positive Control Cinnamic aldehyde	Test Item CADE OIL, rectif. (ex-jun.oxyc)
IC50 (50 % viability)	run 1	49.5 μM	20.8 μg/mL
	run 2	> 64 μM	12.7 μg/mL
	geometric mean	-	16.3 μg/mL
	standard deviation (SD)	-	5.7 μg/mL
IC30 (70 % viability)	run 1	41.7 μM	18.9 μg/mL
	run 2	51.5 μM	10.9 μg/mL
	geometric mean	46.3 μM	14.3 μg/mL
	standard deviation (SD)	7.0 μM	5.6 μg/mL

Table 6. Luciferase activity induction for the test item (compared to the negative control)

conc. [μg/mL]		0.2	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
run 1	rep.1	0.73	0.64	0.76	0.82	1.79	2.10	3.26	0.00	0.00	0.00	0.00	0.00
	rep.2	0.98	0.76	0.94	1.33	1.91	2.62	1.64	0.00	0.00	0.00	0.00	0.00
	rep.3	0.95	0.80	1.14	1.11	2.01	3.01	5.07	0.00	0.00	0.00	0.00	0.00
	mean	0.89	0.73	0.95	1.09	1.90	2.58	3.32	0.00	0.00	0.00	0.00	0.00
	SD	0.14	0.08	0.19	0.26	0.11	0.46	1.71	0.00	0.00	0.00	0.00	0.00
	T- test	0.691	0.054	0.864	0.142	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *
run 2	rep.1	0.73	1.16	1.08	1.30	1.65	2.85	5.32	0.00	0.00	0.00	0.00	0.00
	rep.2	1.08	1.13	1.14	1.35	1.51	2.35	6.97	0.00	0.00	0.00	0.00	0.00
	rep.3	0.95	1.06	1.24	1.31	1.47	2.35	4.10	0.00	0.00	0.00	0.00	0.00
	mean	0.92	1.12	1.15	1.32	1.54	2.52	5.46	0.00	0.00	0.00	0.00	0.00
	SD	0.18	0.05	0.08	0.03	0.09	0.29	1.44	0.00	0.00	0.00	0.00	0.00
	T- test	0.787	0.003 *	0.001 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *	0.000 *

conc. = concentration, rep. = replicate, SD = standard deviation, * - statistical significant (p<0.05)

Table 7. Luciferase activity induction for the positive control (compared to the negative control)

concentration [μM]		4	8	16	32	64
run 1	rep.1	1.20	1.64	3.14	5.97	5.99
	rep.2	1.54	1.65	3.14	6.00	6.86
	rep.3	1.44	1.80	2.36	5.24	8.33
	mean	1.39	1.70	2.88	5.73	7.06
	SD	0.17	0.09	0.45	0.43	1.18
	t-test	0.000*	0.000*	0.000*	0.000*	0.000*
run 2	rep.1	1.40	2.00	2.00	1.54	8.92
	rep.2	1.27	1.51	1.82	2.34	10.18
	rep.3	1.09	1.45	1.75	2.75	7.02
	mean	1.25	1.65	1.86	2.21	8.71
	SD	0.16	0.30	0.13	0.61	1.59
	t-test	0.000*	0.000*	0.000*	0.000*	0.000*

rep. = replicate, SD = standard deviation,* - statistical significant (p<0.05)

Table 8. Imax and EC1.5 values obtained for positive control and test item

		Positive Control Cinnamic aldehyde	Test Item CADE OIL, rectif. (ex-jun.oxyc)
Imax	run 1	7.1	3.3
	run 2	8.7	5.5
	average	7.9	4.4
	standard deviation (SD)	1.2	1.5
EC1.5	run 1	5.4 μM	2.4 μg/mL
	run 2	6.5 .μMl	2.8 .μg/mL
	geometric mean	5.9 μM	2.6 μg/mL
	standard deviation (SD)	0.8 μM	0.3 μg.ml

Table 9. Historical data for positive control (EC 1.5) and negative control (OD600)

	mean	Standard deviation (SD)	mean +2SD	mean -2SD	Minimum	Maximum
Positive Control (mean EC1.5)	21.56	16.12	53.80	-10.69	2.10	59.23
Negative Control (mean OD600)	0.508	0.168	0.844	0.172	0.072	1.161

Table 10. Luminescence reading for the negative (solvent) control.

well no.		1	2	3	4	5	6	7
run 1	rep.1	19735	23464	25297	17035	22867	23250	21483
	rep.2	15616	23489	17676	15203	20048	22048	21598
	rep.3	15131	19948	22887	19034	19461	20508	26354
	mean	20578
	SD	3189
	CV [%]	15.5
run 2	rep.1	32112	37916	37843	37340	39937	38119	34724
	rep.2	35378	37826	38019	36388	40101	36279	32157
	rep.3	32209	38736	36066	37754	34859	28438	28403
	mean	35743
	SD	3354
	CV [%]	9.4

Applicant's summary and conclusion

Interpretation of results:

other: KeratinoSensTM test result is considered 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 can be considered as potentially sensitizing to skin using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A stock solution containing 40 mg/mL test item in DMSO was prepared and used to produce a double dilution series of solutions (12 concentrations). Cinnamic aldehyde is used as positive control in the final concentration range from 4 to 64 μM. The results are compared to negative control (only solvent). Three replicates for each concentrations of test item were used for the luciferase activity measurements and for the cell viability assay. Two independent repetitions (runs) containing each three replicates (i.e. n=6) were performed to derive a prediction (positive or negative). All acceptance criteria (luciferase activity induction and EC1.5 value for positive control, the average coefficient of variation of the luminescence reading for negative control) were within the appropriate range. Therefore, the experiment is considered as valid. Under the experimental conditions, the test item gave positive results in all required criteria in the KeratinoSens assay (luciferase activity induction ≥ 1.5 with viability ≥ 70 %, EC 1.5 < 1000 μM and clear dose-response). The results were consistent in two independent repetitions (runs). Therefore, the test item is positive in this assay and can be considered as potentially skin sensitizing (it can active the Nrf2 transcription factor).