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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb 2018 - 14 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Cymbopogon nardus, ext.
EC Number:
289-753-6
EC Name:
Cymbopogon nardus, ext.
Cas Number:
89998-15-2
IUPAC Name:
Cymbopogon nardus, ext.
Test material form:
liquid
Details on test material:
Citronella nardus oil
CAS no.: 8000-29-1

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: cattle
- Characteristics of donor animals (e.g. age, sex, weight): 6 - 60 months of age
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline without antibiotics in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: no antibiotics used

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C. The corneas were incubated for the minimum of 1 hour at 32°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Yes

SOLVENT CONTROL USED (if applicable)
No

POSITIVE CONTROL USED
Yes

APPLICATION DOSE AND EXPOSURE TIME
Undiluted 750µL, 10 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: yes 120 +/- 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: untill all substance removed

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter (measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA: according to Test Guideline

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
2.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Historical Control Data for the BCOP Studies Feb 2015-2018

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Negative control
- Opacity -2.9 – 3.0 (mean 0.18, SD 1.10, N=113)
- Permeability -0.034 – 0.100 (mean 0.00, SD 0.01, N=113)
- In vitro Irritancy Score -2.8 – 3.0 (mean 0.23, SD 1.13, N=113)

Positive control
- In vitro Irritancy Score 28.0 – 110.9 (mean 55.28, SD 15.14, N=88)

Applicant's summary and conclusion

Interpretation of results:
other: Not irritating to eyes
Remarks:
in accordance with Annex I of the CLP Regulation (1272/2008/EC).
Conclusions:
Citronella nardus oil induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The eye irritation potential of Citronella nardus oil was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test), OECD test guideline 437 under GLP conditions.The negative cotrol, positive control or test item was applied as received, directly on top of the corneas (750 µL) for 10 minutes. Thereafter the eyes were washed and incubated for 120 minutes before evaluating the effects. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 39 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Citronella nardus oil did not induce ocular irritation through both endpoints, resulting in a meanin vitro irritancy score of 2.6 after 10 minutes of treatment. In conclusion, since Citronella nardus oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).