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

Endpoint summary

Description of key information

Key studies presented in this section were conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2019 - 10 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
Description: Clear colourless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

not specified

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin:
EPISKIN™(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-019, Expiry Date: 13 May 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Suggested expiry date: 13 May 2019

Quality Control:
EPISKIN™(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKIN™(SM) Test Kits used in the present study) and are documented in Appendix 2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of test item was added to each skin unit

50 µL of negative control (Physiological saline (0.9% (w/v) NaCl solution)) or positive control (glacial acetic acid) were added to each skin unit

Physiological saline (0.9% (w/v) NaCl solution):
Manufacturer: B. Pharmaceuticals SA
Batch No.: 90352Y05-2
Expiry Date: December 2021
Grade: sterile

Glacial acetic acid:
Supplier: VWR International Ltd.
Batch No.: 17H074111
Expiry date: 05 July 2020

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (23.6-24.7°C)

Duration of post-treatment incubation (if applicable):

After 4 hours incubation time , the EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours
(±1 hour) at 37 °C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light, in a > 95% humidified atmosphere.

At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight (corrosivity part of the test) or for two hours (irritation part of the test) at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Number of replicates:

2 per timepoint

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

129.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

119.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

124.1

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:

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the results. The OD values for the test item treated skin samples showed 124.1% relative viability compared to the negative control.

Validity criteria:
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the blank samples (acidified isopropanol) were 0.046 and 0.047.
The mean OD value of the two negative control tissues was in the recommended range (0.766).
The two positive control treated tissues showed 0.1% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 8.1%
The difference of viability between the two negative control tissue samples in the MTT assay was 2.3%.
High viability results (>>100%) do regularly occur in cases where the test item causes metabolic stimulation in the exposed cells, so the study result is not considered to be invalid.
Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Table 1. Optical Density (OD) and the calculated relative viability % of the samples (Corrosivity test)

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	0.822	0.775	101.2
	2	0.804	0.757	98.8
	Mean	-	0.766	100
Positive Control: Glacial acetic acid	1	0.048	0.002	0.2
	2	0.046	0	0
	Mean	-	0.001	0.1
Test Item: Pentyl Butyrate (Sum of Isomers)	1	1.036	0.99	129.2
	2	0.959	0.912	119.1
	Mean	-	0.951	124.1

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Executive summary:

An in vitro skin corrosivity and irritation test of Pentyl Butyrate (Sum of Isomers) was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in section 3.6). The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

 

Disks of EPISKIN^TM(SM) were treated withthe test itemand incubated for 15 minutes (three units for irritation testing) and 4 hours (two units for corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO_2,in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO₂protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline(0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5% (w/w) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing.For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure withPentyl Butyrate (Sum of Isomers), the mean cell viability was 124.1% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2019 - 10 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
Description: Clear colourless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Source strain:

not specified

Justification for test system used:

The EPISKIN™(SM) model has been validated for corrosivity and irritation testing in an international validation study and its use is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

Human Skin:
EPISKIN™(SM) (Manufacturer: SkinEthic, France, Batch No.: 19-EKIN-019, Expiry Date: 13 May 2019) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin corrosivity and irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Suggested expiry date: 13 May 2019

Quality Control:
EPISKIN™(SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKIN™(SM) Test Kits used in the present study) and are documented in Appendix 2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL of test item were applied evenly to each of three test units
20 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature (24.0-25.2°C)

Duration of post-treatment incubation (if applicable):

EPISKIN™ (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).

After rinsing, the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (±1 hour) at 37 °C in an incubator with 5% CO2, in a > 95% humidified atmosphere.

MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units. The lid was replaced and the plate incubated at 37 °C in an incubator with 5% CO2 for 3 hours, protected from light, in a > 95% humidified atmosphere.

At the end of incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 µL acidified isopropanol (one tube corresponded to one well of the assay plate).

The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight (corrosivity part of the test) or for two hours (irritation part of the test) at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

A blank sample containing 2 mL of acidified isopropanol was processed in parallel in each case.

Number of replicates:

Three replicates per time point were used for test item, three negative controls and three positive controls were also run

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

106

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

87.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

93.8

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:

VIABILITY RESULTS:
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in the results. The mean OD value for the test item treated skin samples showed 93.8% relative viability compared to the negative control.

Validity criteria:
After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the blank samples (acidified isopropanol) were 0.046 and 0.047.

Specific criteria for irritation testing:
The mean OD value of the three negative control tissues was in the recommended range (1.203). Standard deviation of the viability results for negative control samples was 1.7%.
The positive control treated tissues showed 3.4% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.7%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 10.6%.
All these parameters were within acceptable limits and therefore the study was considered to be valid.
Historical control data are presented in Appendix 3.

Table 2: Optical Density (OD) and the calculated relative viability % of the samples (Irritation test)

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	1.264	1.217	101.2
	2	1.226	1.179	98
	3	1.259	1.213	100.8
	Mean	-	1.203	100
Positive Control: Glacial acetic acid	1	0.061	0.034	2.8
	2	0.098	0.051	4.2
	3	0.064	0.038	3.1
	Mean	-	0.041	3.4
Test Item: Pentyl Butyrate (Sum of Isomers)	1	1.322	1.276	106
	2	1.108	1.061	88.2
	3	1.095	1.049	87.2
	Mean	-	1.128	93.8

Notes:

1. Mean blank value was 0.047.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 93.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

Executive summary:

An in vitro skin corrosivity and irritation test of Pentyl Butyrate (Sum of Isomers) was performed in a reconstructed human epidermis model. EPISKIN^TM(SM) is designed to predict and classify the corrosivity and irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in section 3.6). The corrosivity and irritation potential of the test item was evaluated according to the OECD No. 431 and No. 439 guidelines [1, 2].

 

Disks of EPISKIN^TM(SM) were treated withthe test itemand incubated for 15 minutes (three units for irritation testing) and 4 hours (two units for corrosivity testing) at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO₂, in a > 95% humidified atmosphere (irritation testing). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO₂ protected from light, in a > 95% humidified atmosphere. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control) in case of the corrosivity testing. PBS treated epidermis were used as negative control and 5% (w/w) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive control (three units/control) in case of the irritation testing. For each treated tissue, the viability was expressed as a % relative to the negative control. For corrosivity, if the mean relative viability is <35% the test item is considered to be corrosive to skin. For irritation, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with Pentyl Butyrate (Sum of Isomers), the mean cell viability was 93.8% compared to the negative control. This is above the threshold of 50%, thereforethe test item was considered as being non-irritant to skin.

 

The experiment met the validity criteria, therefore the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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).

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.

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.

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

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

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

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.

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

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. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in vitro tests for corrosion and irritation to skin and eye are validated, and the results are suitable for use as the basis for classification according to the UN GHS categories. The results of the tests presented in this section indicate that the registered substance does not meet the criteria for skin irritation/corrosion or eye irritation/corrosion classifications in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).