Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 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 438 (Isolated Chicken Eye 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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

222-081-3

EC Name:

(Z)-3,7-dimethylocta-1,3,6,-triene

Cas Number:

3338-55-4

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Constituent 2

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

1-methyl-4-(prop-1-en-2-yl)cyclohexene

impurity 1

Reference substance name:

2,6-dimethylocta-2,4,6-triene

EC Number:

211-614-5

EC Name:

2,6-dimethylocta-2,4,6-triene

Cas Number:

673-84-7

Molecular formula:

C10H16

IUPAC Name:

2,6-dimethylocta-2,4,6-triene

impurity 2

Reference substance name:

(E)-3,7-dimethylocta-1,3,6-triene

EC Number:

223-241-5

EC Name:

(E)-3,7-dimethylocta-1,3,6-triene

Cas Number:

3779-61-1

Molecular formula:

C10H16

IUPAC Name:

3,7-dimethylocta-1,3,6-triene

Test material form:

liquid

Details on test material:

Name: [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]
Public name: Ocimene PQ
EC numbers: 933-779-9 (pre-registration 222-081-3)
CAS numbers: n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Description: Colourless liquid
Expiry date: 06 June 2019
Purity: Considered as 100%

Specific details on test material used for the study:

Name: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
Public name: Ocimene PQ
EC number: 933-779-9 (pre-registration 222-081-3)
CAS number:n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Appearance: Colourless liquid
Purity:Considered as 100% Expiry date: 06 June 2019
Storage conditions: Room temperature (15-25°C, ≤ 70% relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Animal: chicken
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129, Hungary)
- Age at study initiation : approximately 7 weeks old
- Weight at study initiation: approximately 2.45 kg

ENVIRONMENTAL CONDITIONS
- Transporation: ambient temperature at the earliest convenience

The heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at Citoxlab Hungary Ltd. and processed approximately within 2 hours of collection.

Test system

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL undiluted test item
- Concentration (if solution):

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl) solution.

Name: Physiological saline (Salsol solution, 0.9% (w/v) NaCl)
Batch number:72522Y05-2
Manufacturer: B. Braun Pharmaceuticals SA
Expiry date: 31 May 2020
Storage condition:Room temperature


POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): The positive control eyes were treated with 30 μL of 5% (w/v) Benzalkonium chloride solution

Name:Benzalkonium chloride solution 50% in water
CAS Number: 63449-41-2
Batch number: STBH0549
Expiry date: 31 May 2022
Manufacturer: Sigma-Aldrich Co.
Storage condition:Room temperature

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

NA

Duration of post- treatment incubation (in vitro):

Controls and test eyes: 30, 75,120, 180 and 240 minutes after the post-treatment rinse

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp, with the eye in the head, to ensure that the cornea was not damaged. Only the eyeball with a cornea in good condition was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too
short. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on wet papers in a closed box to mantein the appropriate humidity.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), to avoide too much pressure on the eye by the clamp. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus.
The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the
chamber was closed except for manipulations and examinations, to maintain stable temperature and humidity.
The appropriate number of eyes was selected and carefully checked to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was
measured, any eye with cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. The selected eyes were acclimatize for approximately 45 to 60 minutes at a controlled temperature (32±1.5°C))

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related
effect after treatment. All eyes were considered to be suitable for the assay.

TREATMENTS - APPLICATION DOSE - EXPOSURE TIME - REMOVAL OF TEST SUBSTANCE
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal 30 µL undiluted test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
The positive control eyes were treated in a similar way with 30 µL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl) solution. Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with at least 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

OBSERVATION PERIOD
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. A Haag-Streit Bern 900 slit-lamp microscope,
with depth-measuring device no. 1 and slit-width setting at 9½, was used for the measurements.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

POSITIVE CONTROL USED
Benzalkonium chloride solution 50% in water

METHODS FOR MEASURED ENDPOINTS:
Provided in a separate document - see attachments to this robust study summary

SCORING SYSTEM:
Provided in a separate document - see attachments to this robust study summary

DECISION CRITERIA: According to the OECD guideline

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

up to 75 min

Run / experiment:

1

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

up to 240 min

Run / experiment:

2

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

ca. 0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE Class I

Other effects / acceptance of results:

In the positive control group, severe loosening of epithelium was observed in one eye at 75 minutes after the post-treatment rinse and in the other two eyes at 240 minutes after the post-treatment rinse. No other morphological effect was observed in the study.

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to the result of this study, the substance Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene (Ocimene PQ), EC number: 933-779-9, did not cause eye irritation.

Executive summary:

The irritation effects of the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 were assessed using the Isolated Chicken Eye Test (ICET). The study was performed according to the OECD No. 438 guideline (09 October 2017) and under GLP conditions.

The selected eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL of the test item. The three positive control eyes were treated in a similar way with 30 µL Benzalkonium chloride solution 5% (w/v). The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

The negative control and positive control results were within the historical control data range in the experiment and the quality control standards were met. The experiment was considered to be valid.

The results showed no significant corneal swelling during the four-hour observation period on the test item treated eyes. No significant cornea opacity change was observed in three eyes. No significant fluorescein retention change was noted. No other corneal effect was observed.

In conclusion, the test item Ocimene PQ, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,- triene Ocimene, EC number 933-779-9 is classified as non-irritant, UN GHS Classification: No Category.