Fusanus spicatus, 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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

see Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 142, 31.5.2008, p. 1) as amended B.46

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18.6.2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

EC Number:

204-102-8

EC Name:

5-(2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl)-2-methylpent-2-en-1-ol, stereoisomer

Cas Number:

115-71-9

Molecular formula:

C15H24O

IUPAC Name:

5-(2,3-dimethyltricyclo[2.2.1.0~2,6~]hept-3-yl)-2-methylpent-2-en-1-ol

Constituent 2

Reference substance name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

EC Number:

201-027-2

EC Name:

[1S-[1α,2α(Z),4α]]-2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)-2-penten-1-ol

Cas Number:

77-42-9

Molecular formula:

C15H24O

IUPAC Name:

2-methyl-5-(2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl)pent-2-en-1-ol

Constituent 3

Reference substance name:

(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol

Cas Number:

106-28-5

Molecular formula:

C15H26O

IUPAC Name:

(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol

Constituent 4

Reference substance name:

(Z,6S)-2-methyl-6-(4-methylphenyl)hept-2-en-1-ol

Cas Number:

78339-53-4

Molecular formula:

C15H22O

IUPAC Name:

(Z,6S)-2-methyl-6-(4-methylphenyl)hept-2-en-1-ol

Constituent 5

Reference substance name:

(Z,6R)-2-methyl-6-(4-methylcyclohexa-1,4-dien-1-yl)hept-2-en-1-ol

Cas Number:

698365-10-5

Molecular formula:

C15H24O

IUPAC Name:

(Z,6R)-2-methyl-6-(4-methylcyclohexa-1,4-dien-1-yl)hept-2-en-1-ol

Constituent 6

Reference substance name:

(2R)-6-methyl-2-[(1S)-4-methylcyclohex-3-en-1-yl]hept-5-en-2-ol

Cas Number:

78148-59-1

Molecular formula:

C15H26O

IUPAC Name:

(2R)-6-methyl-2-[(1S)-4-methylcyclohex-3-en-1-yl]hept-5-en-2-ol

Constituent 7

Reference substance name:

(2Z,6S)-2-Methyl-6-(4-methyl-1,3-cyclohexadien-1-yl)-2-hepten-1-ol

Cas Number:

698365-09-2

Molecular formula:

C15H24O

IUPAC Name:

(2Z,6S)-2-Methyl-6-(4-methyl-1,3-cyclohexadien-1-yl)-2-hepten-1-ol

Constituent 8

Reference substance name:

Nerolidol

EC Number:

205-540-2

EC Name:

Nerolidol

Cas Number:

142-50-7

Molecular formula:

C15H26O

IUPAC Name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol

Test material form:

liquid

Details on test material:

Batch no. SS181004

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiDerm™ Reconstructed Human Epidermis, provided by MatTek, Lot.-No. 30804, Keratinocyte strain 00267.
All cells used to produce EpiDerm™ are purchased or derived form tissue obtained form MatTek Corporation from accredited institutions. In all cases, consent was obtained by these institutions from the donor or donor's legal next of kin, for use of the tissues or derivatives of the tissues for research purposes. The cells used to produce EpiDerm™ are screened for potential biological contaminants.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratisla-va.
Designation of the kit: EPI-200-SIT, Day of delivery: 02. Jul. 2019, Batch no.: 30804

Vehicle:

unchanged (no vehicle)

Details on test system:

Test System Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava (Designation of the kit: EPI-200-SIT, Day of delivery: 02. Jul. 2019, Batch no.: 30804).
Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2, Composition see below)
Positive Control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in demineralised water containing 5% SDS. Procured from MatTek In Vitro Life Science Laboratories, batch no.: 032119ALD.
Chemicals and Media
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (= MTT), which can be reduced to a blue formazan. A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL in the freezer (–20 ±5 °C).
2 mL of the stock solution were thawed and diluted with 8 mL of medium. This MTT-solution with the resulting concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium), procured by Life Technologies GmbH, batch no.: 2033578
Assay Medium: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured by MatTek In Vitro Life Science Laboratories, batch no.: 062719MSD
Isopropanol: CH3-CH(OH)-CH3, p.A., 99.9 %, batch no.: 457264070, used as extracting solvent for formazan
DPBS-buffer: Solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared by LAUS GmbH.
Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 031219MSA): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, and H2O ad to 1 L
Composition of the subset from LAUS GmbH (batch no.: 20181022): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, and H2O ad to 1 L
The buffer which was procured by MatTek Corporation was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared by LAUS GmbH was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.
Test Vessels: All vessels used are made of glass or sterilised plastic. The glassware was sterilised before use by autoclaving. The following vessels were used: 6-well-plates, 24-well plates, and 96-well-plate.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL test item (unchanged, no vehicle) were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ±1 °C and 5.0 ±1% CO2 and ≥ 95% relative humidity. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.

Duration of post-treatment incubation (if applicable):

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ±1 °C and 5.0 ±1% CO2 and ≥ 95% relative humidity.

Number of replicates:

two replicates using on each of three tissues.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Absorbance Values

Run / experiment:

test item mean of three tissues

Value:

2.047

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean of three tissues: 2.047

Positive controls validity:

valid

Remarks:

mean of three tissues: 0.061

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

5.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

3.0 %

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

5.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

3.0%

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

5.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

2.9%

Other effects / acceptance of results:

Validity criteria and results are stated as follows:
OD of negative control was found to be 2.0 (validity criterion: ≥0.8 and ≤ 2.8)
% tissue viability of positive control SDS was found to be 3.0% (validity criterion ≤20% of negative control)
SD of mean viability of the tissue replicates (%) were found to be 0.6% (negative control), 0.0% (positive control), and 0.2% (test item) (validity criteria ≤18%)
All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Any other information on results incl. tables

In the following table the outcome of the proficiency chemical testing is stated. All 10 proficiency chemicals were correctly classified.

The demonstration of proficiency was performed under non-GLP conditions but within the GLP-environment at LAUS GmbH.

Results of Proficiency Chemicals

Chemical Name	CAS No.	Physical State	Prediction OECD 439 UN GHS Category	Findings LAUS GmbH
Naphthalene acetic acid	86-87-3	solid	No category	No category
Isopropanol	67-63-0	liquid	No category	No category
Methyl stearate	112-61-8	solid	No category	No category
Heptyl butyrate	5870-93-9	liquid	No category	No category
Hexyl salicylate	6259-76-3	liquid	No category	No category
Cyclamen aldehyde	103-95-7	liquid	Category 2	Category 2
1-bromohexane	111-25-1	liquid	Category 2	Category 2
Potassium hydroxide (5% aq.)	1310-58-3	liquid	Category 2	Category 2
1-methyl-3-phenyl-1-piperazine	5271-27-2	solid	Category 2	Category 2
Heptanal	111-71-7	liquid	Category 2	Category 2

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The mean value of relative tissue viability of the test item was reduced to 5.7% after the treatment. This value is below the threshold for skin irritation (50%). Therefore, the test item is considered as at least irritant to skin.

Executive summary:

Three tissues of the human skin model EpiDerm™ were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤2.8, OD was 2.0.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.0% (required: ≤20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 5.7%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin.

Therefore, the test item Fusanus spicatus, ext. is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.