Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 June 2018 - 07 November 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)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

268-417-2

EC Name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

68084-04-8

Molecular formula:

C13H19N

IUPAC Name:

3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Constituent 2

Reference substance name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

244-530-2

EC Name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

21690-43-7

Molecular formula:

C13H19N

IUPAC Name:

4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Test material form:

liquid

Specific details on test material used for the study:

Name: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile
Public name: Azuril
EC number: 915-371-2
CAS number: n/a (pre-registration 21690-43-7)
Batch/Lot number: A170421E
Appearance: Clear, pale yellow liquid
Purity**: 99.35 %
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:

DETAILS ON SOURCE CHICKENS FOR HEADS/EYES
- Source: Slaughter house TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129, Hungary)
- Age during the head collection: 7 weeks
- Weight during the head collection: about 2.65 kg
- Collection of heads: by a slaughter house technician
- Transportation of heads to laboratory: within 2 hours of collection (in plastic box wrapped with tissue paper moistened with saline)
- Acclimatization period and temperature: approximately 45 to 60 minutes at 32 ± 1.5°C
- Temperature during testing: 32 ± 1.5°C

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL of test item, positive and negative control

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes (evaluations after 30, 75, 120, 180 and 240 minutes post-teatment)

Number of animals or in vitro replicates:

3 eyes for test item
3 eyes for positive control
1 eye for negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes selection: After removing the head from the plastic box, it was put on soft paper. 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 fluorescein-treated cornea was examined with a hand-held slit lamp with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes: 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. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. 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 so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
Baseline assessments: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 minute 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.

NUMBER OF REPLICATES
3 eyes for test item, 3 eyes for positive control, 1 eye for negative control

NEGATIVE CONTROL USED
0.9% (w/v) NaCl

POSITIVE CONTROL USED
5% (w/v) Benzalkonium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
30 μL of test item, positive and negative control. Exposure 10 seconds.

OBSERVATION PERIOD
240 minutes (evaluations after 30, 75, 120, 180 and 240 minutes post-teatment)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed thoroughly with 20 mL physiological saline solution at ambient temperature
- Indicate any deviation from test procedure in the Guideline : no deviations

METHODS FOR MEASURED ENDPOINTS:
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. No histopathology was performed.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: According to the OECD 438 test guideline.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Run / experiment:

1

Value:

-1.1

Vehicle controls validity:

not applicable

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:

at up to 240 min

Run / experiment:

1

Value:

-2.2

Vehicle controls validity:

not applicable

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

Remarks:

mean maximum

Run / experiment:

1

Value:

0.5

Vehicle controls validity:

not applicable

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

Remarks:

mean

Run / experiment:

1

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE Class II

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation in the isolated chicken eyes test with Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile], the test item is non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (09 October 2017).

After the zero reference measurements, the 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 undiluted test item. The three 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). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

No significant corneal swelling (0 to -5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. Slight fluorescein retention change (severity 0.5 on two eyes and severity 1 on one eye) was noted in all three eyes. No other corneal effect was observed.

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

Based on this in vitro eye irritation in the isolated chicken eyes test with Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile], the test item is non-irritant, UN GHS Classification: No Category.