Juniper, Juniperus mexicana, ext., isomerized, acetylated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-01-2015 to 21-05-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Test material

Test material information

Constituent 1

Reference substance name:

Juniper, Juniperus mexicana, ext., isomerized, acetylated

EC Number:

293-297-3

EC Name:

Juniper, Juniperus mexicana, ext., isomerized, acetylated

Cas Number:

91053-33-7

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

(1S,2R,5S,7S)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene; 1-[(1R,2R,5S,7R)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-en-9-yl]ethan-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: At room temperature protected from light.
- Other: Pale yellow

Test animals / tissue source

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): > 9 months old (typically).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. Post-excision, the isolated eyes were stored in physiological saline in the cooled slaughter-house until transportation to the laboratory using a suitable container. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: < 24 hours. Corneas were prepared for testing immediately on same day arrival.
- indication of any existing defects or lesions in ocular tissue samples: None. Only corneas free from damage utilised (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches were discarded).
- Indication of any antibiotics used: None reported.

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): 0.75 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against an air 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.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically (e.g. presenting defects such as vascularization, pigmentation, opacity and scratches) and where necessary discarded. Additionally, only corneas with opacity < 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline solution

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium Chloride solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes at 32 ± 1ºC.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 ± 10 minutes. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: Opacity, Permeability and In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n=3)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

IVIS scores ranged from 1.0 to 1.3 after 10 minues treament

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None. No pH effect of the test item was observed on the rinsing medium.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. 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.
- Acceptance criteria met for positive control: Yes. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 128.2 and was within the historical positive control data range.
- Range of historical values if different from the ones specified in the test guideline: The mean in vitro irritancy score of the negative control were less than the upper limits of the laboratory historical range and for the positive control (10% (w/v) Benzalkonium Chloride) at 128.2 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Applicant assessment of the data indicates that the positive control and negative controls were within the current historic control range (HCD) (documented in the full study report), each meeting the validity criteria respectively.

Any other information on results incl. tables

Table 1 : Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity #1	Mean Permeability #1	Mean In vitro Irritation Score #1 , 2
Negative Control	0	0.000	0.0
Positive Control (Benzalkonium chloride)	81.7	3.102	128.2
Test item	1.0	0.010	1.1

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2 : Opacity score

Eye	Opacity before treatment	Opacity after treatment	Final opacity #1	Negative control corrected final opacity #2	Mean Opacity
Negative Control
1	0	0	0	0	0
2	0	0	0	0
3	0	0	0	0
Positive Control
4	0	83	84	84	81.7
5	0	83	84	84
6	0	76	77	77
Test item
13	0	1	1	1.0	1.0
14	-1	0	1	1.0
15	-1	0	1	1.0

#1 Final Opacity = Opacity after treatment – Opacity before treatment

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Table 3 Permeability score individual values (uncorrected)

Eye	Dilution Factor	OD490 1	OD490 2	OD490 3	Average OD490	Final OD	Mean Final Negative Control
	Negative Control
1	1	0.010	0.011	0.011	0.011	0.011	0.008
2	1	0.004	0.004	0.004	0.004	0.004
3	1	0.009	0.007	0.008	0.008	0.008
	Positive Control
4	6	0.314	0.311	0.312	0.312	1.874
5	6	0.482	0.479	0.473	0.478	2.868
6	6	0.794	0.781	0.775	0.783	4.700
	Test item
13	1	0.022	0.033	0.024	0.026	0.026
14	1	0.009	0.011	0.012	0.011	0.011
15	1	0.013	0.013	0.018	0.015	0.015

 

Table 4 : In vitro irritancy score

Eye	Negative control corrected final opacity	Negative control corrected Final OD490	In vitro Irritancy Score #1
Negative Control
1	0.0	0.003	0.0
2	0.0	-0.004	-0.1
3	0.0	0.000	0.0
Positive Control
4	84.0	1.829	111.4
5	84.0	2.823	126.3
6	77.0	4.655	146.8
Test item
13	1.0	0.019	1.3
14	1.0	0.003	1.0
15	1.0	0.007	1.1

#1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test item is not considered to be irritant in the in vitro eye corrosion/irritation test using Bovine Corneal Opacity and Permeability model. The in vitro irritancy score (IVIS) was < 3.0 in the prediction model.

Executive summary:

The study was performed according to OECD TG 437 and EU Method B.47 to assess the eye irritancy potential in accordance with GLP of the item in isolated bovine corneas. The purpose of this test was to evaluate the test item for its potential to induce serious eye damage/ocular irritation. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. A total of three corneas per treatment group were used. A volume of 750 microlitres of the item was placed the cornea. The negative control group received saline and the positive control group received 10% benzalkonium chloride. For each group the corneas were incubated for 10 minutes at 32 degrees C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Eagle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 (10% (w/v) Benzalkonium Chloride) was 128.2 (GHS Category 1 : eye damage prediction) and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment. Based on these results the item is considered to be not irritating or corrosive in the Bovine Corneal Opacity and Permeability test.