Isobutyl isobutyrate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

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

Test system:

human skin model

Source species:

human

Cell type:

other: human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen

Cell source:

other: A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum from an adult human.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface

Duration of treatment / exposure:

Tissues were treated with the test item for an exposure period of 15 minutes

Duration of post-treatment incubation (if applicable):

42-Hour post-exposure incubation period

Number of replicates:

Triplicate tissues were treated with the test item.

Details on study design:

Pre-Test Procedure
To identify this possible interference, the test item is checked for the ability to directly reduce MTT.

Pre-incubation (Day 0: Tissue Arrival)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main test
Application of Test Item and Rinsing (Day 1)
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Irritation / corrosion parameter:

% tissue viability

Value:

110.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

other: The test item was classified as non-irritant. The test item is not requiring classification for skin irritancy under either UN GHS or EU CLP.

Conclusions:

The test item was classified as non-irritant. The test item is not requiring classification for skin irritancy under either UN GHS or EU CLP.

Executive summary:

The skin irritation potential of the Isobutyl isobutyrate was evaluated using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the post-exposure incubation period tissues were taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made for extraction of formazan crystals. At the end of the formazan extraction period the optical density was measured at 570 nm. The percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) was calculated. The relative mean viability of the test item treated tissues was 110.6% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The relative mean viability of negative control was 100% and for positive control was 8.5%.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Deviations:

no

Principles of method if other than guideline:

PRINCIPLE OF THE TEST METHOD

The test material is applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit- 24-well plate

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

Tissues were treated with the test item for exposure periods of 3 and 60 minutes.

Number of replicates:

Two

Details on study design:

Pre-Incubation
0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods.
EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing

Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure.For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. 25 mg of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

103.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

non-corrosive to the skin

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute

Value:

101.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

non-corrosive to the skin

Interpretation of results:

other: No category. Not requiring classification to UN GHS or EU CLP

Conclusions:

The test item is not corrosive to skin and not classified for corrosivity in UN GHS or CLP regulations.

Executive summary:

The corrosivity potential of Isobutyl isobutyrate was evaluated using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues samples were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in isopropanol for MTT extraction. At the end of the formazan extraction period the optical density (OD) was measured at 570 nm. Percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) of test item for 3-minute exposure was 103.2% and after 60 minutes was 101.4%. Positive control showed the viability of 4.8 and 5.2% in 3 and 60 minutes exposure. The test item is not corrosive to skin and not classified for corrosivity in UN GHS or CLP regulations.

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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

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

GLP compliance:

yes

Specific details on test material used for the study:

The test substance is identified as Isobutyl isobutyrate. The purity is 98.99%. The physical state/appearance of material is clear colorless liquid. The expiry date of material is 06 February 2018. The substance can be stored at room temperature in the dark conditions.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF BOVINE EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.

The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer
(Annex 1). The average opacity for all corneas was calculated. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.650 g of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

APPLICATION OF SODIUM FLUORESCEIN
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

PERMEABILITY DETERMINATIONS
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of 98.99% purity

Duration of treatment / exposure:

test item was applied for 10 minutes followed by an incubation period of 120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCEY SCORE
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATIONS
The condition of the cornea was visually assessed post treatment.

DATA INTERPRETATION
The test item was classified according to the following prediction model:

IVIS CLASSIFICATION
below 3 no category. Not requiring classificatio to UN GHS or EU CLP
between 3 and 55 No prediction of eye irritation can be made
greater than 55 Category 1 (UN GHS or EU CLP (causes serius eye damage)

CRITERIA FOR AN ACCEPTABLE TEST
For an acceptable test the following positive control criterion should be achieved:

20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2016 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3.

For an acceptable test the following negative control criteria should be achieved:

Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.4 and for permeability ≤0.072.

REFERENCE ITEM PREPARATION
The negative control item, sodium, chloride 0.9% w.v, was used.
The positive control item, ethanol, was used with >99.8% purity.

Irritation parameter:

in vitro irritation score

Value:

3

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: No category. Not requiring classification to UN GHS or EU CLP

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

Executive summary:

The purpose of this test was to identify test item Isobutyl isobutyrate can induce serious eye damage and to identify test item not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The undiluted test item was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).The IVIS scores for the test item, the negative control and the positive control were 3.0, 1.3, and 33.0, respectively. The test item is not requiring classification for eye irritancy under either UN GHS or EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The test article is not irritating to eyes or skin and does not requiring classification for under either UN GHS or EU CLP.