Juniper, Juniperus oxycedrus, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 October 2019 - 04 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Remarks:

EpiDerm™ model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Justification for test system used:

The EpiDerm™ model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-218-SIT)
- Tissue batch number(s): 30829
- Production date: 30.09.2019
- Delivery date: 01.10.2019
- Date of initiation of testing: 01.10.2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37ºC for 35 min and room temperature for 25 min.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15-times with DPBS at 1-minute-intervals
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2, 90 RH
- Spectrophotometer: Microplate reader: FLUOstar Omega (BMG Labtech)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: linear range of 0.0 – 3.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.82 (specification 1< OD < 3). Historical negative control mean OD range = 0.883-2.960.
- Barrier function: 6.45 h (Specification 4.77h < ET50< 8.72h)
- Morphology: appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermedaite spinous and granular layers.
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: 2 freeze-killed control tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin (or corrosive) if the viability after 60 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL (47 μL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

13.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: in the pre-test for assessment of direct reduction of MTT by the test item, the MTT solution turned blue/purple, so the test item was presumed to have reduced the MTT and additional control (NSMTT) was performed. However, the relative viability of tissue treated with the test item was ≤ 50 % compared to the negative control (positive result) and thus the true tissue viability calculation was not necessary.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed and the 10 of 10 proficiency substances were correctly classified.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 6.7% (acceptablility criteria, SD ≤ 18%) and OD mean was 1.423 (acceptability criteria, ≥ 0.8 and ≤ 2.8).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.4% (acceptablility criteria, SD ≤ 18%) and mean viability was 3.1% (acceptability criteria, < 20 %).
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 2.3% (acceptablility criteria, SD ≤ 18%).

Any other information on results incl. tables

Table 1. Blank corrected absorbance values (OD 570 nm)

OD 570 nm		Negative control		Positive control		Test item: CADE OIL, RECTIF. (EX-JUN.OXYC)
Tissue number	Measurement replicate	OD 570 nm	Mean	OD 570 nm	Mean	OD 570 nm	Mean
1	A	1.531	1.543	0,042	0,051	0,185	0,179
	B	1.555		0,059		0,174
2	A	1.341	1.342	0,040	0,039	0,171	0,168
	B	1.343		0,038		0,165
3	A	1.390	1.384	0,042	0,042	0,235	0,236
	B	1.378		0,041		0,237
Mean of the three tissues		1,423		0.044		0.194

Table 2. Tissue viability [%]

	Negative control	Positive control	Test item: CADE OIL, RECTIF. (EX-JUN.OXYC)
Tissue no. 1 viability	108.4	3.6	12.6
Tissue no. 2 viability	94.3	2.7	11.8
Tissue no. 3 viability	97.2	2.9	16.6
Mean Tissue viability	100	3.1	13.7
SD (±)	6.7	0.4	2.3
%CV	6.7	12.4	16.7

Table 3. Historical data of absorbance values for negative and positive controls

	Negative Control DPBS buffer	Positive Control 5 % SDS
Mean OD 570 nm	2.038	0.042
Standard deviation (SD)	0.437	0.007
Minimum	0.883	0.029
Mean -SD	1.164	0.028
Maximum	2.960	0.059
Mean +2SD	2.912	0.056

In above table the means of OD 570 nm for the negative controls and positive controls of all performed experiments up to 04. Oct. 2019 are stated and compared with the values which were found in this study.

Applicant's summary and conclusion

Interpretation of results:

other: classified as corrosive (Cat 1) or irritant (Cat 2) (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item is considered to be irritating / corrosive to skin (category 1 or 2) in the Reconstructed human Epidermis (RhE) Test Method.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human EpiDerm™ model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 30 μL test item for 60 minutes. Exposure of the test item was terminated by washing the tissues 15-times with DPBS at 1-minute-intervals. The tissues were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT for 3 hours at 37 ºC, 5 % CO2 and 90 RH, extracting the precipitated formazan crystals using isopropanol during 2 hours at room temeprature, and measuring the concentration of formazan by determining the absorbance (OD at 570 nm). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. All acceptance criteria (absorbance value for negative control, mean value of relative tissue viability of positive control, variation within the tissue replicates) were within the appropriate range. Therefore, the experiment was considered as valid. After the treatment with the test item, the mean value of relative tissue viability was reduced to 13.7 %. This value is below the threshold for skin irritation potential (≤ 50%). Therefore, the test item is considered as skin irritant (cat 2) or skin corrosive (Cat 1).