Registration Dossier

Administrative data

Description of key information

Two in vitro studies (skin & eye) and one in vivo study (eye) concluded that the test material was non-irritating to skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

DETERMINATION OF SKIN IRRITATION POTENTIAL USING THE SKINETHIC RECONSTRUCTED HUMAN EPIDERMAL MODEL

Introduction. The purpose of this study was to determine the skin irritation potential of the test item using the SkinEthic Reconstructed Human Epidermal model (RHE, SkinEthic Laboratories, Nice, France) following treatment periods of 4 and 24 hours. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Methods. The experimental design of the study consists of a test for direct reduction of MTT by the test item, followed by the main test. For the main test, triplicate SkinEthic tissues were treated with approximately 25 mg of test item and exposed for 4 hours and 24 hours. The tissues were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure times. Duplicate untreated tissues were used for each exposure period to serve as negative controls. Duplicate tissues treated with 50 µL of Triton X-100 (0.1% w/v in distilled water) were used for the 24-Hour exposure period to serve as a positive control. At the end of the 4-Hour exposure period each SkinEthic tissue was rinsed using Dulbecco's Phosphate Buffered Saline (DPBS) and placed into a 'holding plate' until all the tissues had been rinsed. The rinsed tissues (2 per group) were then transferred to an MTT 'loading plate' and incubated at 37°C for 3 hours in a humidified atmosphere of 5% CO2 in air. At the end of this time, each SkinEthic tissue was blotted dry and placed into an MTT 'extraction plate' in order to extract all of the reduced MTT from the tissues. The remaining test item treated tissue was retained for possible histology. The same rinsing, loading, extraction and retention procedures were repeated for the 24-Hour tissues once the 24-Hour exposure period was complete. The maintenance medium in each well of the 24-Hour exposure period treatment plates were retained for possible analysis ofinflammatory mediator levels of IL-1α and IL-8.

At the end of the extraction period, the extracted MTT solution was mixed for each SkinEthic tissue and 3 x 200 µL samples representing each tissue were transferred to the appropriate wells of a 96 well plate. The optical density at 540nm (OD540) of each well was measured. Data are presented in the form of percentage viability (MTT conversion relative to negative controls) for each of the two exposure periods. The results were used in order to make a prediction of skin irritation potential.

Results. The relative mean viability of the test item treated tissues was 108.6% after the 4-Hour exposure period and 99.5% after the 24-Hour exposure period. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators.

Conclusion. The test item was considered to be a Non-Irritant. GHS: Considered not to be a significant irritant.

DETERMINATION OF EYE IRRITATION POTENTIAL USING THE SKINETHIC RECONSTRUCTED HUMAN CORNEAL EPITHELIAL MODEL

Introduction. The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic Reconstructed Human Corneal model (RHC, SkinEthic Laboratories, Nice, France) following treatment periods of 10 and 60 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. The experimental design of the study consists of a test for Direct Reduction of MTT by the test item, followed by the main test. For the main test, triplicate SkinEthic tissues were treated with the test item and exposed for 10 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure period.

Duplicate tissues served as the negative and positive controls for each exposure period.

At the end of the 10-Minute exposure period each SkinEthic tissue was rinsed using Dulbecco's Phosphate Buffered Saline (DPBS) and placed into a 'holding plate' until all the tissues had been rinsed. The rinsed tissues (two per group) were then transferred to an MTT 'loading plate', and incubated at 37°C for 3 hours in a humidified atmosphere of 5% CO2 in air. The remaining test item treated tissue was retained for possible histology. Following MTT loading, each SkinEthic tissue was blotted dry and placed into an MTT 'extraction plate' in order to extract all of the reduced MTT from the tissues. The same rinsing, retention, loading and extraction procedures were repeated for the 60-Minute tissues once the 60-Minute exposure period was complete.

At the end of the extraction period, the extracted MTT solution was mixed for each SkinEthic tissue and 3 x 200 µL samples, representing each tissue, were transferred to the appropriate wells of a 96 well plate. The optical density at 540nm (OD540) of each well was measured. Data are presented in the form of percentage viability (MTT conversion relative to negative controls) for each of the two exposure periods. The results were used to make a prediction of the eye irritation potential of the test item.

Results. The relative mean viability of the test item treated tissues was 97.5% after a l0-Minute exposure period and 88.7% after a 60-Minute exposure period. It was considered unnecessary to proceed with tissue histology.

Conclusion. Due to an equivocal result a Rabbit Enucleated Eye Test was conducted. The results of both studies were combined to determine labelling requirements and were considered to be as follows: Not classified in EU - Mild to moderate irritant.

RABBIT ENUCLEATED EYE TEST

Introduction. A study was performed to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test item in man.

Methods. 0.1 mL of the test item, which was found to weigh approximately 83 mg, was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Results. Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity

Fluorescein Uptake

Corneal Swelling (%)

Condition of Corneal Epithelium

Test Eyesa

Control Eyes*b

Cldy x Area

Int x Area

60 mins

120 mins

240 mins

60 mins

120 mins

240 mins

0

0

9.1

14.7

15.7

5.2

3.9

0.2

Normal

 

Conclusion. Following assessment of the data for all endpoints the test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Justification for classification or non-classification

Eye irritancy potential

Due to an equivocal result a Rabbit Enucleated Eye Test was conducted. The results of both studies were combined to determine labelling requirements and were considered to be as follows: Not classified in EU - Mild to moderate irritant. Furthermore, following assessment of the data for all endpoints the test item was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Skin Irritation Potential

The test item was considered to be a Non-Irritant. GHS: Considered not to be a significant irritant.