Fusanus spicatus, ext.

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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)

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

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

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.