Reaction mass of Phenol, dodecyl-, sulfurized, calcium salts and benzene and butene and calcium carbonate and calcium dihydroxide and sulphur trioxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with current Guidelines and GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The SkinEthic HCE model assesses the irritation potential of a test item by examining the cytotoxic effects of the test item after a defined exposure
period and recovery time.

The endpoint of the HCE assay is the estimation of cell viability by assaying the reduction of MTT by mitochondrial enzymes. Irritant materials are
identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

EC Number:

903-161-3

IUPAC Name:

Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, reaction products with sulphur monochloride and decene, reaction products with polybutenyl benzenesulphonic acid, carbon dioxide and calcium hydroxide

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): EC-903-161-3
- Physical state: Liquid
- Expiration date of the lot/batch: 19 December 2013
- Storage condition of test material: In the dark at ambient temperature

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST SYSTEM
- HCE Irritation Assay
- SkinEthic HCE Tissues

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Mean Application rate: 60 µL/cm2

Duration of treatment / exposure:

60 mins.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The HCE tissues were rinsed with PBS (25 mL) then the upper surface of the HCE® tissue was lightly swabbed with a cotton swab.
- Time after start of exposure: 60 mins.

SCORING SYSTEM: The test item was considered to be an irritant to eyes if the tissue viability after exposure and post-treatment incubation was less than or equal to (=) 50% of the negative control value.

TOOL USED TO ASSESS SCORE: Absorbance Measurement after Formazan extraction.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

107.09

Remarks on result:

no indication of irritation

Remarks:

viability of 107.09% of the negative control value

In vivo

Irritant / corrosive response data:

Exposure to EC-903-161-3 resulted in a mean HCE viability of 107.09% of the negative control value.

Any other information on results incl. tables

EC-903 -161 -3

Non-viable controls

The mean absorbance value of the three replicate non-viable tissues dosed with EC-903-161-3 was 0.221 ± 0.041. The mean absorbance value of the three replicate undosed non-viable tissues was 0.029 ± 0.005. Therefore, the absorbance value for the effect of the unremoved test item was 0.192. This value was subtracted from the absorbance value of the viable tissues dosed with EC-903-161-3.

 

Viable controls

The results were similar for the three viable HCE units dosed with EC-903-161-3. Exposure to EC-903-161-3 resulted in a mean HCE viability of 107.09% ± 21.20% of the negative control value.

Negative Control

The results were similar for the three viable HCE tissues dosed with the negative control. Exposure to Dulbecco’s PBS resulted in a mean HCE viability of 100.00% ± 16.12%.

Positive Control

The results were similar for the three viable HCE tissues dosed with the positive control. Exposure to Triton X-100 solution (0.2%, w/v) resulted in a mean HCE viability of 5.19% ± 2.45%.

Histology Results

Microscopic analysis of cross sections of HCE^®tissues treated with EC-903-161-3, and PBS showed little or no difference in tissue integrity for the two treatments. It was not possible to prepare stained cross sections for the positive control. Therefore, the study conclusion is based solely on the tissue viability results.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: The test item was considered to be an irritant to eyes if the tissue viability after exposure and post-treatment incubation was less than or equal to (=) 50% of the negative control value.

Conclusions:

In conclusion, EC-903-161-3 was demonstrated to be non irritant to eyes when tested in vitro using the HCE® reconstructed human corneal model.

Executive summary:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of EC-903-161-3 was assessed using the HCE in vitro ocular irritation test.

 

Eye irritation potential was assessed by applying EC-903-161-3 (30 µL) to the exposed surface of three viable HCE tissues for 60 min. The surface area of the HCE was 0.5 cm², therefore the application rate was 60 µL/cm². After the 60 min exposure period, the test item was washed from the surface of the HCE using Dulbecco’s phosphate-buffered saline (PBS; 25 mL) and cotton swabs. The HCE tissues were then incubated for a recovery period of 16 h in a humidified incubator set to maintain temperature and CO₂levels of 37°C and 5%, respectively. Following incubation, the HCE tissues were transferred to maintenance medium containing MTT (0.5 mg/mL) and returned to the incubator for3 h. The HCE tissues were then transferred to isopropanol in order to extract the formazan. After extraction (90 min), the formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control, Triton X-100 solution (0.2%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual HCE tissue was calculated as a percentage of the mean negative control viability (defined as 100%). Due to the intrinsic ability of EC-903-161-3 to reduce MTT to formazan (demonstrated under Charles River Study No. 791169) it was necessary to employ non-viable control tissues to account for the effect of the non-specific reduction.

 

In addition to the tissues used for the viability assessment, one HCE tissue was dosed for each treatment as above and prepared for histological analysis. Cross-sections of each tissue were formalin-fixed, paraffin embedded and stained by the standard haematoxylin-eosin method. The tissues were then examined microscopically for tissue damage.

 

Exposure to EC-903-161-3 resulted in a mean HCE viability of 107.09 ± 21.20% of the negative control value. Exposure to the positive control, Triton X-100 (0.2%, w/v), resulted in a mean HCE viability of 5.19 ± 2.45% of the negative control value. Cell viability values below a threshold of 50% of the negative control viability indicate that the test item is irritant.

 

Microscopic analysis of cross sections of HCE tissues treated with EC-903-161-3, and PBS showed little or no difference in tissue integrity for the two treatments. It was not possible to prepare stained cross sections for the positive control. Therefore, the study conclusion is based solely on the tissue viability results.

 

In conclusion, EC-903-161-3 was demonstrated to be non-irritant to eyes when tested in vitro using the HCE reconstructed human corneal model.