Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2019 - 16 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl butyrate

EC Number:

208-739-2

EC Name:

Pentyl butyrate

Cas Number:

540-18-1

Molecular formula:

C9H18O2

IUPAC Name:

pentyl butyrate

Constituent 2

Reference substance name:

2-methylbutyl butyrate

EC Number:

256-973-9

EC Name:

2-methylbutyl butyrate

Cas Number:

51115-64-1

Molecular formula:

C9H18O2

IUPAC Name:

2-methylbutyl butyrate

Test material form:

liquid

Specific details on test material used for the study:

Name: Pentyl Butyrate (Sum of Isomers)
Description: Clear colourless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity).

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Source:
TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

Age and collection:
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.

Storage conditions:
After collection, 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 within 2 hours of collection.

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 or slit lamp microscope, 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 eyeball 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 the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time:
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). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. 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 temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. 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. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Baseline assessment:
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 were observed in all eye. 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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 position, while the test material was applied onto the centre of the cornea. 30 µL of the 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).

Duration of treatment / exposure:

The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 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.

Duration of post- treatment incubation (in vitro):

The control eyes 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.

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes, and one negative control eye were examined during the study.

Details on study design:

Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum at up to 75 min

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean maximum at up to 240 min

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Mean maximum

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.17

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 observations: NONE
Overall ICE Class: 3xI
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Any other information on results incl. tables

Test item:

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.60%	I
Mean maximum corneal swelling at up to 240 min	0.60%	I
Mean maximum corneal opacity change	0.33	I
Mean fluorescein retention	0.7	I
Other Observations	None
Overall ICE Class	3 x I

Based on this in vitro eye irritation study in isolated chicken eyes with Pentyl Butyrate (Sum of Isomers), the test item is non-irritant, UN GHS Classification: No Category. 

Positive control:

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.90%	II
Mean maximum corneal swelling at up to 240 min	27.70%	III
Mean maximum corneal opacity change	4	IV
Mean fluorescein retention	3	IV
Other Observations	Severe loosening of epithelium was observed in two eyes at 180 minutes and in one eye at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

Based on these observations, the positive control substance (Benzalkonium chloride solution (5% (w/v)) was classified as severe irritant according to the EU regulations. GHS Classification: Category 1.

Negative control:

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity change	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3 x I

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Historical Control data (updated on 07 November 2018):

Negative Control: Physiological Saline

Observation	Min value	Max value
Mean maximum corneal swelling at up to 75 min	-3.2%	3.4%
Mean maximum corneal swelling at up to 240 min	-4.8%	3.4%
Mean maximum corneal opacity change	0.00	0.50
Mean fluorescein retention	0.00	0.50
Number of studies	416

Positive Control: 5% (w/v) Benzalkonium chloride solution

Observation	Min value	Max value
Mean maximum corneal swelling at up to 75 min	-8.5%	27.0%
Mean maximum corneal swelling at up to 240 min	-10.7%	38.3%
Mean maximum corneal opacity change	2.50	4.00
Mean fluorescein retention	1.50	3.00
Number of studies	234

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria for “No category” (all true): 3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II: True No severe corneal morphological changes: True Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse: True

Conclusions:

Based on these in vitro eye irritation assays in isolated chicken eyes with Pentyl Butyrate (Sum of Isomers), 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 (25 June 2018).

 

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 exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 µL 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.

 

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 experiment. Thus, the study was considered to be valid.

 

No significant cornea swelling change (mean ≤ 5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5 was on two eyes and no significant cornea opacity change on one eye) was observed. No significant fluorescein retention change (severity 0.5 on one eye and no significant cornea opacity change on two eyes) was noted. No other corneal effect was observed.

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:	TRUE
No severe corneal morphological changes:	TRUE
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	TRUE
Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	FALSE
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	FALSE
Corneal opacity = 4 at any time point (in at least 2 eyes):	FALSE
Severe loosening of epithelium (in at least 1 eye):	FALSE
Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	FALSE
Particles of test item were stuck to the cornea and could not be washed off during the study:	FALSE

Based on these in vitro eye irritation assays in isolated chicken eyes with Pentyl Butyrate (Sum of Isomers), the test item is non-irritant, UN GHS Classification: No Category.