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Eye irritation

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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:
23 DEC 2016 to 08 FEB 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene
EC Number:
207-491-2
EC Name:
[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene
Cas Number:
475-20-7
Molecular formula:
C15H24
IUPAC Name:
4,8,8-trimethyl-9-methylenedecahydro-1,4-methanoazulene
Test material form:
liquid
Specific details on test material used for the study:
Longifolene Coeur BLO (mono-constituent)
Appearance: Pale yellow liquid
Batch no: H2225-02
Expiry date: 11/04/2019
Storage conditions: ambient (5-45 C)
Purity: 84.05%

Impurity: (1R,4E,9S)-4,11,11-trimethyl-8-methylide nebicyclo[7.2.0]undec-4-ene

Negative control:
Physiological Saline NaCl 0.9% (w/v)
Supplier: Eurovet, Bladel, the Netherlands
Appearance: Clear Liquid
Batch no: H2225-02
Expiry date: 06/2019
Storage conditions: ambient temperature

Positive control:
Benzalkonium Chloride (BAC)
Appearance: Clear colorless liquid
Concentration: 50% (aqueous)
Lot no.: SZBF0150V
Supplier: Sigma-Aldrich
CAS Reg. no.: 63449-41-2
Expiry date: 01/10/2020
Storage conditions: Ambient temperature
Concentration to be tested: 5% (v/v) aqueous

Test animals / tissue source

Species:
chicken
Details on test animals or tissues and environmental conditions:
Eye donors from ROSS spring chickens
Body weight: 1.5-2.5 kg
Age: ~ 7 weeks
Sex: Male and female

Heads of the chickens were obtained from poultry slaughterhouse v.d. Bor, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Single application of 30 µL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
0, 30, 75, 120, 180 and 240 minutes after treatment
Number of animals or in vitro replicates:
Negative control: 1 eye sample
Positive control: 3 eye samples
Test substance: 3 eye samples
Details on study design:
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (Triskelion, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32˚C (water pump set at 36.4˚C; Lauda 103, Germany).
After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope, set at 0.095 mm. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.
Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluores-cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.
At time t = 0 (i.e. immediately after the zero reference measurement), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards.
Next, the eyes (corneas) were treated with the study substances 30 µL for 10 seconds followed by rinsing with 20 mL of saline.

After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment. Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 µm and stained with PAS
(Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
fluorescein retention score
Remarks:
30 minutes
Run / experiment:
Eye numbers 1, 2, 3
Value:
ca. 0
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
0/30/75/120/180/240 minutes
Run / experiment:
Eye numbers 1, 2, 3
Value:
ca. 0
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
corneal swelling 
Remarks:
180 minutes
Run / experiment:
Mean of eye number 1, 2, 3
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
corneal swelling 
Remarks:
240 minutes
Run / experiment:
Mean of eye number 1, 2, 3
Value:
ca. 0
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
corneal swelling 
Remarks:
120 minutes
Run / experiment:
Mean of eye number 1, 2, 3
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
corneal swelling 
Remarks:
75 minutes
Run / experiment:
Mean of eye number 1, 2, 3
Value:
3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
corneal swelling 
Remarks:
30 minutes
Run / experiment:
Mean of eye number 1, 2, 3
Value:
3
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

An overall summary of the results of the positive control and test substance based on maximum mean values for corneal swelling, corneal opacity, and fluorescein retention, the irritation categories assigned to the parameters, the irritation index and the regulatory classifications are presented in Table 1.
Longifolene Coeur BLO caused no or very slight corneal swelling (mean of 3%).
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.


 




































Test Material



Maximum mean score for:



Irritation


categories1



Irritation index2



Classifications (EU-CLP3/UN-GHS4)



Swelling %



Opacity



Fluorescein retention



Longifolene Coeur BLO



3



0



0



I;I;I



3



NC/NC



BAC 5%



34



3.05



3.0



IV;IV;IV



154



1/1



TABLE 1


1 I = no effect; II = slight effect; III = moderate effect; IV = severe effect.


2 Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)


3 EU-CLP: NC = not classified; Category 2 = Irritating to eyes; Category 1 = irreversible effects on the eye/serious damage to the eye. Regulation (EC) No 1272/2808 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2806.


4 UN-GHS: NC = not classified; Category 2B = mild irritant, causes eye irritation; Category 2A = irritant, causes eye irritation; Category 1 = irreversible effects on the eye/serious damage to the eye. United Nations-Economic Commission for Europe. (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2007.


5 Severe loosening of epithelium


 


The individual and mean values for corneal swelling, corneal opacity and fluorescein retention recorded at the various observation time points for the test substance are presented in Table 2.


































































































































































































































































Eye No. 1



Corneal thickness (instrument units)



Corneal Opacity



Fluorescein Retention



T (min)= ->



-45



0



30



75



120



180



240



0



30



75



120



180



240



0



30



 



64



62



65



65



64



63



62



0



0



0



0



0



0



0



 



swelling %



 



 



4.8



4.8



3.2



1.6



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Eye No. 2



Corneal thickness (instrument units)



Corneal Opacity



Fluorescein Retention



T (min)= ->



-45



0



30



75



120



180



240



0



30



75



120



180



240



0



30



 



63



62



64



64



63



63



62



0



0



0



0



0



0



0



0



swelling %



 



 



3.2



3.2



1.6



1.6



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Eye No. 3



Corneal thickness (instrument units)



Corneal Opacity



Fluorescein Retention



T (min)= ->



-45



0



30



75



120



180



240



0



30



75



120



180



240



0



30



 



64



63



64



63



63



62



62



0



0



0



0



0



0



0



0



swelling %



 



 



1.6



0



0



0



0



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



 



Mean



 



 



3



3



2



1



0



Mean



0



0



0



0



0



Mean



0



TABLE 2

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Applying the classification criteria of the ICE, the following irritation classifications can be assigned:
Longifolene Coeur BLO: Not Classified (UN-GHS and EU-CLP classifications).

Executive summary:

Longifolene Coeur BLO was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control
(BAC 5%). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 µL for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.
Longifolene Coeur BLO only caused very slight corneal swelling (mean of 3%).
Microscopic examination of the corneas did not reveal any abnormalities.
The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.
The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane.
Applying the classification criteria of the ICE, the following irritation classifications can be assigned:
Longifolene Coeur BLO: Not Classified (UN-GHS and EU-CLP classifications).