Benzene, di-C10-14-alkyl derivs., sulfonated, sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

EpiOcular™ Cornea Epithelial Model

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 12, 2016 to December 23, 2016

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:

2017

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)

Deviations:

yes

Remarks:

regarding the study plan but the study integrity was not adversely affected by the deviations

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, di-C1 0-14-alkyl derivs., sulfonated, sodium salts

IUPAC Name:

Benzene, di-C1 0-14-alkyl derivs., sulfonated, sodium salts

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Benzene, di-C1 0-14-alkyl derivs., sulfonated, sodium salts
- ZS name: ALKYLBENZOLNACHLAUF, SULFONIERT, NA-SALZ
- ZS number: 1344
- Batch: SEALS 2011-104-5-8
- Appearance, colour: brown clear liquid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test substance to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 minutes

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

Number of animals or in vitro replicates:

3

Details on study design:

The test consists of application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 minutes. After exposure the cornea epithelial construct is thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 hours incubation period determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement - optical density reading at 570 nm). Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: cell viability (%)

Remarks:

experiment 1

Run / experiment:

reduction of MTT

Value:

>= 43 - <= 65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

cell viability was spread over two categories

Results 2

Irritation parameter:

other: cell viability (%)

Remarks:

experiment 2 (repeat)

Run / experiment:

reduction of MTT

Value:

>= 55 - <= 77

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

cell viability was spread over two categories

Other effects / acceptance of results:

- The test substance showed colour interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test substance was 0.43% of the negative control tissues. True tissue viability was calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT.
- Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues was 54%. Since the relative tissue viability for the test substance was spread over two categories (65 and 43%, respectively). The experiment was repeated.
- In this repeat experiment, the non-specific colour of the test substance was 1.00% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test substance compared to the negative control tissues was 66%. Again, the relative tissue viability for the test substance was spread over two categories (55 and 77%, respectively).
- The positive control had a mean cell viability of 15% and 4% after 30 ± 2 minutes exposure, in the initial and repeat experiment, respectively. The absolute mean OD570 values (optical density at 570 nm) of the negative control tissues were within the acceptability range from > 0.8 to < 2.5.
- In both tests equivocal results were obtained after 30 minutes treatment. Therefore, no conclusion could be made about the eye hazard potential of the test substance in the EpiOcular™ test under the experimental conditions described in this report.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, no conclusion could be made about the eye hazard potential of the test substance in the EpiOcular™ test.

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the test substance according to OECD Guideline 492 (EpiOcular™ Cornea Epithelial Model), in compliance with GLP. The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. The test consisted of an application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 min. After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 h incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement - optical density reading at 570 nm).  Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The test substance showed colour interference in aqueous conditions. In addition to the normal procedure, two tissue were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test substance was 0.43% of the negative control tissues. True tissue viability was calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 54%. Since the relative tissue viability for the test substance was spread over two categories (65 and 43%, respectively). The experiment was repeated. In the repeat experiment, the non-specific colour of the test substance was 1.00% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 66%. Again, the relative tissue viability for the test substance was spread over two categories (55 and 77%, respectively). In both tests equivocal results were obtained after 30 min treatment. The positive control had a mean cell viability of 15 and 4% after 30 ± 2 min exposure, in the initial and repeat experiments, respectively. The absolute mean OD570 values (optical density at 570 nm) of the negative control tissues were within the acceptability range from > 0.8 to < 2.5. The tests functioned properly. Under the study conditions, no conclusion could be made about the eye hazard potential of the test substance (Verbaan, 2017).