Registration Dossier

Administrative data

Endpoint:
eye irritation
Remarks:
other: In vitro assay to examine ocular irritation of a test item using the SkinEthic HCE® model.
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.

The SkinEthic reconstructed Human Corneal Epithelium (HCE) model has undergone ECVAM prevalidation testing and has been shown to have a high correlation with existing in vivo and in vitro data for known eye irritants and non-irritant compounds.

GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naphthoxyethanol
- Physical state: solid
- Expiration date of the lot/batch: July 2013
- Storage condition of test material: In the dark at ambient temperature
- Lot/batch No.: E00173-186

Results and discussion

In vivo

Results
Remarks on result:
other: Exposure to 2-Naphthoxyethanol resulted in a mean HCE viability (corrected for effect of unremoved test item) of 2.13% of the negative control value. 2-Naphthoxyethanol was demonstrated to be irritant to eyes when tested in vitro.
Irritant / corrosive response data:
Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13% of the negative control value.

Any other information on results incl. tables

MTT Direct Reduction Test

The test was scored by visual assessment of the formation of purple-coloured formazan. The positive control (eugenol) reduced the MTT solution to purple-coloured formazan. The negative control (water) did not reduce MTT to formazan. The results of the MTT direct reduction test showed that Naphthoxyethanol did reduce MTT to formazan.

Skin Ethic HCE Irritation Test

The results of the ocular irritation assay with Naphthoxyethanol are shown in Table 1 (relative viability of the HCE tissues expressed as a percentage of the mean negative control value).

 

Table 1

Treatment

Mean Relative
Viability per
Tissue (%)

Mean Relative
Viability per
Treatment (%)

SD (%)

Naphthoxyethanol (Corrected)

2.32

2.13

0.17

2.06

2.00

Triton X-100 (0.2%, w/v)

(Positive Control)

3.48

6.54

4.01

11.08

5.06

PBS Solution

(Negative Control)

109.34

100.00

8.12

96.04

94.62

 

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% ± 8.12%.

 

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 6.54% ± 4.01%.

 

Naphthoxyethanol

Non-Viable Controls

The mean absorbance value of the three replicate non-viable tissues dosed with Naphthoxyethanol was 0.010 ± 0.002. The mean absorbance value of the three replicate undosed non-viable tissues was 0.045 ± 0.007. Therefore, a negative absorbance value was obtained for the effect of the unremoved test item and the calculations did not require to be adjusted.

 

Viable Tissues

The results were similar for the three viable HCE units dosed with Naphthoxyethanol. Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13% ± 0.17% of the negative control value.

 

Histology Result

Microscopic analysis appeared to show some damage to the tissues dosed with Naphthoxyethanol. However, due to the preparation error which resulted in the lack of images for the positive control, the decision was made to reject the results of the histology and to base the conclusion of the study solely on the tissue viability results. This decision is justified, as the histology is an add-on and is not required for interpretation of irritation potential. Therefore, the integrity of the study is not compromised by the rejection of the histology data.

 

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, Naphthoxyethanol was demonstrated to be 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 Naphthoxyethanol was assessed using the HCE in vitro ocular irritation test.

 

Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, which is a measure of cell viability in the assay. The results of the MTT direct reduction test showed that Naphthoxyethanol did reduce MTT to formazan. Therefore, non‑viable controls were tested in parallel with the irritation test to quantify this effect.

 

Eye irritation potential was assessed by applying Naphthoxyethanol (ca 30 mg) to the exposed surface of three HCE tissues for 60 min. Prior to applying the test item, the HCE surface was pre‑moistened with sterile, ultra‑pure water (30 µL). The surface area of the HCE was 0.5 cm2, therefore the application rate was ca 60 mg/cm2. After the 60 min exposure period, the test item was washed from the surface of the HCE using Dulbecco’s phosphate-bufferedsaline (PBS; 25 mL) and cotton swabs. Additional aliquots of PBS were required to remove all the applied Naphthoxyethanol, which had adhered to the tissue surface. 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 for 3 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%).

 

In addition to the three tissues assessed for viability, a fourth 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. Due to a preparation error, the results of the histological analysis were rejected as it was not possible to evaluate the positive control. Therefore, the study conclusion was based solely on the tissue viability results. The integrity of the study was unaffected as the histological evaluation isan add‑on and is not required for interpretation of irritation potential. There was little visual difference between the test and negative control samples.

Exposure to Naphthoxyethanol resulted in a mean HCE viability of 2.13 ± 0.17% of the negative control value. Exposure to the positive control, Triton X-100 (0.2%, w/v), resulted in a mean HCE viability of 6.54 ± 4.01% 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.

 In conclusion, Naphthoxyethanol was demonstrated to be irritant to eyes when testedin vitrousing the HCE reconstructed human corneal model.