Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by organic solvents extraction

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August - 19 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

27 April 2017

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by organic solvents extraction

EC Number:

946-442-6

Molecular formula:

Not applicable

IUPAC Name:

Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by organic solvents extraction

Test material form:

solid

Remarks:

brown to brown-red paste

Details on test material:

- Name (as cited in study report): LABDANUM RESINOID / F
- Storage condition of test material: room temperature, darkness, closed container

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Justification for test system used:

Following the REACH bottom-up strategy described in the ECHA R.7a guidance, the SkinEthic™ Reconstructed Human Epidermis Model method was used to assess skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ Reconstructed Human Epidermis Model, SkinEthic Laboratories, Lyon, France (RHE/S/17)
- Tissue batch number(s): 17-RHE-091
- Expiration date: September 11, 2017
- Date of testing: between 31 August 2017 and 07 September 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 41 hours and 15 minutes at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 9130617).
- Observable damage in the tissue due to washing: none, but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 hours at 37°C
- Spectrophotometer: ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
- Wavelength: 570 nm
The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.2 (CV = 1.9%) (O.D. > 0.7)
- Barrier function: 4.8 h (4.0h < ET50 < 10.0 h)
- Morphology: 6 cells layers (> 4). Absence of significant histological abnormalities. Satisfactory (Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum)
- Contamination: absence of HIV1 & 2 antibodies, hepatitis C and antibodies, hepatitis C and antigen HBsp on blood; absence of mycoplasma on epidermal cells
- Reproducibility: All of the values for the negative and positive control groups fell within or very close to the historical ranges of the testing laboratory obtained in the previous year. This was taken to show the correct functioning of the test system.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding approximately 50 mg of the test item applied on a nylon mesh to 1 mL of solution of MTT at 1 mg/mL. A yellow to brown solution was observed after 3 hours of incubation between 36.5°C and 37.8°C, 5% CO2.
> Therefore, there is no direct interaction between the test item and MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive or irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is < 50% and in the absence of information on a skin corrosion test.
- The test substance is considered to be non-irritant to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 % in distilled water

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 25 minutes post-incubation period at 37°C, 5% CO2

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1

Value:

58.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

1.6%

Remarks on result:

not determinable

Remarks:

1/3 epidermis has a% viability < 50%

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

2

Value:

9.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

2.3%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- In the first run, the mean percent viability of the treated tissues was 58.9%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).
- In the second run, the mean percent viability of the treated tissues was 9.4%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate).

- OTHER EFFECTS:
- Visible damage on test system: none but residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse
- Direct-MTT reduction: none.
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

ACCEPTANCE OF RESULTS:
FIRST RUN:
As the percentage of viability of 1/3 of RHE are ≤ 50% it’s not possible to conclude for the test item.
The acceptability criteria defined in OECD 439 are not respected. A second run is necessary.
SECOND RUN:
- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.623 and 1.226). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.]
- Acceptance criteria met for positive control: yes, the positive control is classified as irritant
- Acceptance criteria met for variability between replicate measurements: no, the SD value of the % viability for the negative control is > 18 (31.2%). However, this deviation has no impact of the overall conclusion of the study. Other SDs values are ≤ 18% (values between 2.3and 9.4%).
- Range of historical values if different from the ones specified in the test guideline: TO BE COMPLETED

Any other information on results incl. tables

Table 7.3.1/1: First run - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.573	0.613	0.759	80.8	100.0	16.7
		0.626
		0.641
	2	0.818	0.824		108.6
		0.827
		0.826
	3	0.826	0.839		110.6
		0.831
		0.860
Positive control	1	0.012	0.013	0.012	1.7	1.6	0.1
		0.013
		0.013
	2	0.011	0.011		1.4
		0.011
		0.011
	3	0.013	0.012		1.8
		0.011
		0.011
Test item	1	0.102	0.102	0.447	13.4	58.9	39.8
		0.102
		0.103
	2	0.576	0.574		75.7
		0.559
		0.586
	3	0.729	0.665		87.7
		0.609
		0.657

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Table 7.3.1/12: Second run - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.629	0.628	0.919	68.3	100.0	31.2
		0.633
		0.623
	2	0.843	0.929		101.1
		1.007
		0.938
	3	1.126	1.201		130.6
		1.190
		1.189
Positive control	1	0.026	0.025	0.021	2.7	2.3	0.3
		0.025
		0.024
	2	0.021	0.019		2.1
		0.019
		0.018
	3	0.024	0.020		2.2
		0.020
		0.018
Test item	1	0.079	0.080	0.087	8.7	9.4	2.0
		0.079
		0.084
	2	0.077	0.073		7.9
		0.073
		0.070
	3	0.111	0.107		11.6
		0.107
		0.105

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (corrosive) or Category 2 (irritant) base don GHS criteria

Conclusions:

Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the SkinEthic reconstructed human epidermis model.

As the test item LABDANUM CTE RES SUPER INDE was a paste considered as a solid, it was administered after being directly applied on a nylon mesh (corresponding to 16 mg) in order to cover the entire surface of 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 41 hours 15 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean percent viability of the treated tissues was 58.9%, versus 1.6% in the positive control (5% Sodium Dodecyl Sulfate).

As the percentage of viability of 1/3 of RHE are ≤ 50% it’s not possible to conclude for the test item. The acceptability criteria define in OECD 439 are not respected. A second run was necessary.

During the second run, the mean percent viability of the treated tissues was 9.4%, versus 2.3% in the positive control (5% Sodium Dodecyl Sulfate).

Concerning acceptability criteria of this second run:

- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is > 0.4 and < 1.5 (values between 0.623 and 1.226). [The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol : the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.]

- Acceptance criteria met for positive control: yes, the positive control is classified as irritant

- Acceptance criteria met for variability between replicate measurements: no, the SD value of the % viability for the negative control is > 18 (31.2%). However, this deviation has no impact of the overall conclusion of the study. Other SDs values are ≤ 18% (values between 2.3and 9.4%).

The quality criteria required for acceptance of results in the test were therefore considered to be satisfied.

 

Under the experimental conditions of this study, in the absence of information on skin corrosion, the test substance has to be classified in Category 2 "Irritating to skin" or Category 1 "Corrosive" according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.