Methyl 4-[2-[4-(5-methyl-2-benzoxazolyl)phenyl]vinyl]benzoate

Endpoint summary

Description of key information

Not skin irritant

Not eye irritant

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

SKIN IRRITATION

EpiSkinTM SM test of the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 28 July 2015.

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1h) in an incubator with 5±1 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item showed no ability to become coloured in contact with water. However, the test item has an intrinsic colour (yellowish), two additional test item-treated tissues were used for the non-specific OD evaluation.

Under the tested conditions, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 85 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin.

The skin irritation potential of the substance was also investigated on 2 rabbits. 0.5 ml of substance diluted at 0, 10 and 1 % were applied to each 2.5 x 2.5 cm patch. In the case of undiluted test item application very slight, barely perceptible erythema, mild edema in both rabbits were observed. In the case of test item at 10 % well-developed erythema on the scarified flank skin of a rabbit was recorded, while not scarified skin showed very slight, barely perceptible erythema and edema. In the case of the test item at 1 % very slight, barely perceptible erythema in both rabbits were seen and very slight, barely perceptible edema on the scarified flank skin of one rabbit was observed.On the basis of the above described findings, the substance has been indicated as slightly irritating to the skin in all 3 tested concentrations.

Although the test can be considered as sufficientely documented and meeting generally accepted scientific principles, it cannot be considered as acceptable for assessment because the content of Fluorescent Brightener 371 in the lot tested was very limited. Therefore, it has been here reported only for completeness sake.

EYE IRRITATION

The eye irritating potential of Fluorescent Brightener 371 was investigated by Isolated Chicken Eye Test (ICET). The test compound was applied in a single dose of 30 mg/eye. Three test item treated eyes and three positive control eyes were used. One negative control eye was treated with 30 µl saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 ml saline solution at ambient temperature and this procedure was repeated for each eye.

Adherence of the test item and the positive control Imidazole was observed on the cornea surfaces at 240 min after the post-treatment rinse. Test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 1xI, 2xII. Positive and negative controls showed the expected results. The experiment was considered to be valid, but unfortunately, no prediction can be made, according to the guideline OECD 438.

For this reason an additonal in vitro study was carried out to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model, according to the OECD testing guideline 492.

Before treatment the tissues were pre-wetted with approximately 20 μl of Ca2+ Mg2+ Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 mg/units) test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2 °C in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca2+ Mg2+ Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37 ± 2 °C in an incubator with 5 ± 1 % CO2 protected from light, ≥ 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

The test item showed no ability to become coloured in contact with water. However, it showed highly opacity solution after contact with isopropanol, so it was tested for its ability to absorb significantly light at the wavelength (570 nm) used for the MTT determination. According to the results of the OD measurement the use of additional color controls was necessary.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 71 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. Thus, the test item is considered as non-irritant to eye.

An old test is also available; the eye irritation potential of the substance was investigated on 2 rabbits. 0.1 ml of the test item diluted at 0, 10 and 1% was once applied in the conjunctival sac of the eye. The application of undiluted test item causedvery slight weakening a 1/2 or 3/4 of the total corneal surface, clearly injected vessels or diffuse carmine redness without distinction of individual vessels, slight swelling in a rabbit, leaving secretion in both rabbits.In the case of test item at 10 % very slight cloudiness on 1/4 of the entire corneal surface, clearly injected vessels, slight swelling and low secretions in both rabbits were recorded.vIn the case of test item at 1 % very slight turbidity on 1/4 or 1/2 of the entire cornea surface was observed; in addition, clearly injected vessels and low secretion in both rabbits were recorded. On the basis of the above described findings, the undiluted and 10 and 1 % diluted substance was described as slightly irritating to the mucous membrane.

Although the test can be considered as sufficientely documented and meeting generally accepted scientific principles, it cannot be considered as acceptable for assessment because the content of Fluorescent Brightener 371 in the lot tested was very limited. Therefore, it has been here reported only for completeness sake.

Justification for classification or non-classification

According the results obtained in the in vitro studies carried out on the test substance in order to investigate both skin and eye irritation/corrosion potential, and according to the classification criteria set up on the relative test guidelines and in line with the principle of the CLP Reagulation No 1272/2008, the substance under investigation was not classified.