Registration Dossier

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 guidelineopen allclose all
Qualifier:
according to
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:
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
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

Resultsopen allclose all
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.

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.