Isobutylbenzene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Isobutylbenzene

EC Number:

208-706-2

EC Name:

Isobutylbenzene

Cas Number:

538-93-2

Molecular formula:

C10H14

IUPAC Name:

(2-methylpropyl)benzene

Test material form:

liquid

Details on test material:

- Analytical purity: 99.9%
- Lot/batch No.: EXP-18/11/346

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: EXP-18/11/346
- pH value: ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

Test animals / tissue source

Species:

human

Strain:

other: normal human derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

Description of the cell system used: Reconstructed human cornea-like epithelium
- Model used: EpiOcular™ model (OCL-200)
- Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue batch number: 27004
- Certificate of authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
Tissue viability: acceptance criteria met
Barrier function: acceptance criteria met
Sterility: no contamination

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Deionized water, sterile

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume): 50 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume): 50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2

Details on study design:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were immersed and swiveled three times in each of three beakers filled with sterile PBS.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
A direct interference was not observed.

NUMBER OF REPLICATE TISSUES PER TEST CHEMICAL AND CONTROLS: 2

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm, without reference filter

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to the eye if the mean relative tissue viability with a test material is less than 55%.
- The test substance is considered to be non- irritant to the eye if the mean relative tissue viability with a test material is greater than 65%.
- In case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability of 55 - 65%, a second test should be considered as well as a third one in case of discordant results between the first two tests.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 492: The „borderline“-evaluation (60 ± 5%) was determined statistically using historic data of the test facility and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

ACCEPTANCE CRITERIA
- Negative control: Tissue viability is acceptable if the mean OD570 of the negative control (NC) is > 0.8. The mean OD570 of the NC should not exceed 2.5.
- Positive control: Methyl acetate used as positive control (PC) usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is < 20%.

HISTORICAL DATA (see table 1)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean viability (%)

Run / experiment:

1st run

Value:

105

Vehicle controls validity:

other: = negative control

Negative controls validity:

not valid

Remarks:

mean OD570 was exceptionally low

Positive controls validity:

valid

Remarks on result:

other: invalid

Remarks:

criterion for inter-tissue variability of the test-substance treated tissues was not met

Results 2

Irritation parameter:

other: Mean viability (%)

Run / experiment:

2nd run

Value:

77

Vehicle controls validity:

other: = negative control

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Potential compound residues remained on the tissues treated with the test substance in the 2nd test run. However, that did not interfere with the MTT assay as the test substance was not able to reduce MTT directly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (2nd run only)
- Acceptance criteria met for positive control: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

Any other information on results incl. tables

Table 1: Historic control data

Historic Range of NC
OD570
Protocol	Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Feb 2016 - Jul 2017	1.845	0.349	2.544	1.146
Protocol for solids	Feb 2016 - Jul 2017	1.678	0.268	2.213	1.142

 

Historic Range of PC
OD570
Protocol	Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Feb 2016 - Jul 2017	0.534	0.166	0.867	0.201
Protocol for solids	Feb 2016 - Jul 2017	0.313	0.112	0.537	0.088

 

Viability (%)
Protocol	Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
Protocol for liquids	Feb 2016 - Jul 2017	29.2	8.8	46.7	11.7
Protocol for solids	Feb 2016 - Jul 2017	18.4	5.3	29.1	7.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed for the EpiOcular Test alone, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in the current case the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Two test runs were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after

exposure/post-incubation by using a colorimetric test.

The test substance was not able to reduce MTT directly.

Results of the 1st test run:

As the acceptance criterion for inter-tissue variability of the test-substance treated tissues was not met (relative viability values for single tissues: 82.5% and 127.5%) and the mean OD570 of the negative control (NC) was exceptionally low (0.801), a 2nd experiment was performed.

Results of the 2nd test run:

The relative mean viability of the tissues treated with the test substance was 77%. All acceptance criteria were met. Potential compound residues remained on the tissues treated with the test substance in the 2nd test run. However, that did not interfere with the MTT assay as the test substance was not able to reduce MTT directly.

Based on the results observed in the EpiOcular Test alone, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.