Fusanus spicatus, ext.

Endpoint summary

Description of key information

Under the conditions of an in vitro study for eye irritation/damage (OECD 437), the test item Fusanus spicatus, ext. showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.49 and accordingly, the substance is considered non-irritant to eyes according to CLP (Regulation EC No 1272/2008).

 

Under the conditions of an in vitro study for skin corrosion/ irritation (OECD 439) the test item Fusanus spicatus, ext. is considered as at least irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 5.7%. This value is below the threshold for skin irritation (50%) and thus, the substance is to be classified as a skin irritant, category 2 according to CLP (Regulation EC No 1272/2008).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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 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.

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

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 %

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%

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.

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

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.47

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name Fusanus spicatus, ext.
Batch no. SS181004
Appearance clear, mustard-yellow liquid
Composition Main components: Z-α-santalol; epi-α-bisabolol; Z-β-santalol; epi-β-santalol; Z-α-trans-bergamotol; E,E-farnesol; Z-nuciferol; Z-lanceol
Purity not applicable, UVCB
Homogeneity homogeneous
Expiry date 16. Jul. 2021

Species:

other: eyes from Bos primigenius Taurus (fresh bovine corneas)

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 10 minutes.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the appropriate liquid (test substance, negative control and positive control, in triplicates) were added through the refill hole in the anterior holder on the corneas

Duration of treatment / exposure:

Exposure time of the controls and test item on the corneas was 10 minutes at 32 ±1 °C.

Duration of post- treatment incubation (in vitro):

After thorough rinsing the anterior chambers with cMEM (complete MEM) with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phenol red and the cornea holders were stored for additional 2 hours at 32 ±1 °C (post-incubation).

Number of animals or in vitro replicates:

test item, negative control and positive control were tested in triplicates each.

Irritation parameter:

in vitro irritation score

Run / experiment:

1. Measurement

Value:

2.67

Vehicle controls validity:

not applicable

Remarks:

substance was applied neat

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2. Measurement

Value:

2.36

Vehicle controls validity:

not applicable

Remarks:

substance was applied neat

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3. Measurement

Value:

2.45

Vehicle controls validity:

not applicable

Remarks:

substance was applied neat

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three independent experiments

Value:

2.49

Vehicle controls validity:

not applicable

Remarks:

substance was applied neat

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity: According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.
The validity criteria and findings are given as follows:
Mean IVIS of negative control HBSS ≤ 3 (criterion) was found 0.36 and thus, is valid
Mean IVIS of positive control (DMF undiluted) in historical control range (54.01 - 141.57) was found as 110.04 and thus, considered valid.
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
Assessment:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Classification Scheme
IVIS ≤ 3  No category according to UN GHS
IVIS > 3 and ≤ 55  No prediction can be made according to UN GHS
IVIS > 55  Eye damage Category I according to UN GHS
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.
The test item Fusanus spicatus, ext. showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was 2.49.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Opacity and Permeability Values

Theilluminance(unit: LUX) values which were measured before and after exposure are given in the following table:

Table1-aIlluminance Values

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	997	1031	1041	1047	1042	1036	1025	1024	993
(I) Measured values after exposure	991	1025	1030	980	979	972	339	320	326

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation:

Table1-b Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.72	2.30	1.90
Opacity after exposure	3.98	2.54	2.34
Opacity Difference	0.26	0.24	0.44
Mean Opacity Difference	0.32

Rep. = Replicate

 

Table1-c Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	1.66	1.86	2.10	2.54	2.58	3.90
Opacity after exposure	4.47	4.51	4.83	87.46	94.99	92.52
Opacity Difference	2.81	2.66	2.73	84.91	92.41	88.62
Opacity Difference corrected	2.49	2.34	2.42	84.60	92.09	88.30
Mean Opacity Difference corrected	2.42			88.33

Rep. = Replicate

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Table 1-d Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.034
2. Measurement	0.036
3. Measurement	0.035
Mean	0.035

 

Table1-e Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measure-ment	0.045	0.036	0.036	0.051	0.040	0.041	1.207	1.346	1.886
2. Measure-ment	0.044	0.033	0.034	0.049	0.039	0.041	1.198	1.359	1.856
3. Measure-ment	0.045	0.033	0.034	0.049	0.038	0.038	1.210	1.356	1.945
1. Measure-ment – blank	0.0100	0.0010	0.0010	0.0160	0.0050	0.0060	1.1720	1.3110	1.8510
2. Measure-ment – blank	0.0090	-0.0020	-0.0010	0.0140	0.0040	0.0060	1.1630	1.3240	1.8210
3. Measure-ment – blank	0.0100	-0.0020	-0.0010	0.0140	0.0030	0.0030	1.1750	1.3210	1.9100
Mean of each replicate	0.0097	-0.0010	-0.0003	0.0147	0.0040	0.0050	1.1700	1.3187	1.8607
Mean of the 3 replicates	0.0028			--			--
Corrected	--	--	--	0.0119	0.0012	0.0022	1.1672	1.3159	1.8579
Corrected mean of the 3 replicates	--			0.0051			1.4470

Rep. = Replicate

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table9.2‑a      IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.41	0.36	31.32%
	0.23
	0.44
Test Item Fusanus spicatus, ext.	2.67	2.49	6.43%
	2.36
	2.45
Positive Control DMF undiluted	102.11	110.04	6.55%
	111.83
	116.17

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study (OECD 437), the test item Fusanus spicatus, ext. showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.49 and accordingly, the substance is considered non-irritant to eyes according to CLP (Regulation EC No 1272/2008).

Executive summary:

This in vitro study was performed to assess the corneal damage potential ofFusanus spicatus, ext.by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test itemFusanus spicatus, ext.was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

 

The test item was tested neat.

Under the conditions of this test, the test itemFusanus spicatus, ext.showed no effects on the cornea of the bovine eye. The calculated mean IVIS was 2.49.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

 

The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.

 

No observations were made which might cause doubts concerning the validity of the study outcome. No deviations from the study plan were observed. The test is considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Under the conditions of an in vitro study for eye irritation/damage (OECD 437), the test item Fusanus spicatus, ext. showed no effects on the cornea of the bovine eye and accordingly, the substance is considered non-irritant to eyes according to CLP (Regulation EC No 1272/2008). Under the conditions of an in vitro study for skin corrosion/ irritation (OECD 439) the test item Fusanus spicatus, ext. is considered as irritant to skin and thus, the substance is to be classified as a skin irritant, category 2 according to CLP (Regulation EC No 1272/2008).