2-[(2-nitrophenyl)amino]ethanol

Endpoint summary

Description of key information

Based on the results of the two key studies (Klimisch 1, GPL, OECD 438 & 492 guideline methods) , the test article HC Yellow No. 2 was classified as Category 2A for Eye Irritation H319 "Causes serious eye irritation".

According to the key studies (Warren, 2018, Klimisch 1, GLP, OECD TG 431 & 439), the test article HC Yellow No. 2 was not classified for skin irritation according to CLP criteria.

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:

adverse effect observed (irreversible damage)

Additional information

Two available key studies were performed in order to assess the eye irritation potential of the registered substance and one study was available to assess skin irritation potential of the test item :

- An Isolated Chicken Eye Test (ICE) was performed following OECD GLP principle, OECD guideline 438 method and was quoted as Klimisch 1 (Prinsen 2016):

HC Yellow No.2 was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (Sodium Hydroxide). Chicken

eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse.

After the exposure procedure, a yellow staining of the cornea was observed. The intensity of the staining decreased over time and had cleared at 120 min after administration. At sampling of the corneas for histopathological examination, a yellow staining of the vitreous body was observed. HC Yellow No. 2 (B041; GTS119138) caused slight or moderate corneal swelling (mean swelling 17%), moderate opacity (mean score 2.0) and moderate fluorescein retention (mean score 2.0).

-A Human Cornea Model Test was performed following OECD 492 method and GLP principle and was quoted as Klimisch 1 (Duschl 2017):

Each 50 mg of the test item, were applied to each of duplicate EpiOcularTM tissues for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also

applied to duplicate tissues each. Viability quantification was performed on cells using MTT reduction to formazan approach after 6 hours exposure period and 18 hours post exposure period. Irritating effects were observed following incubation with HC Yellow No. 2 (B041). Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased to 2.5% (threshold for irritancy: < 60%).

Two other in vivo studies were available for eye irritation assessment (Burnett, Supporting study, Klimisch 3, 1987) :

Neat 100 mg in the first study, and 0.1 mL (diluted at 10%) of HC Yellow No. 2 in the second study were instilled in the conjunctival sac of the left eye of four New Zealand White rabbits. The right eye was untreated. The eyes of one half of the rabbits were rinsed with 20 ml distilled water 20 seconds after treatment and scored 1 hour and 1, 2, and 3 days post instillation according to the Draize method. The test item induced mild irritant effect but reversible. However, the study method had some deficiencies: two animals were only exposed to 20 seconds (instead of 1 hours required) and the two others animal were exposed continuously until the end of exposure period (3 animals required in the guideline).In the second study, the dose applied was not sufficient according to REACh requirements.

- A in vivo skin irritation test was performed and was quoted as Klimisch 2 : Five hundred milligrams of HC Yellow n° 2 were applied as aqueous slurry to the nonoccluded, intact skin of the backs of six New Zealand White rabbits and left in contact for 24 hours. The sites were scored for dermal irritation at 24 and 72 hours post test material application according to the Draize Method. No apparent dermal irritation was produced by HC Yellow n° 2. Under the experimental conditions of the study, the test item HC Yellow No. 2 did not induce irritation to rabbit skin after 24 hours of skin contact. The method of the study was in accordance with Draize method (publised in 1965 and citated in OECD guideline 404), the study was considered as relevant for assessment. 24 hours exposure period performed instead of 4 horus required in current guideline was acceptable. Hence, the test substance was not classfifed for skin irritation.

-An in vitro skin corrosion test was performed and was quoted as Klimisch 1 (Warren, 2018, GLP): This study was performed according to OECD 431 TG method. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes.  Negative and positive control groups were treated for each exposure period.  The test item was found to have the potential to cause color interference with the MTT end-point therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT assay to determine the percent cellular viability. The test item did not induced adverse effect to test system, the cellular viability valuues were 93.8% and 110.2%, for respectively, 3 and 60 minutes exposure period.

-An in vitro skin irritation test was performed and was quoted as Klimisch 1 (Warren, 2018, GLP) : This study was performed according to OECD 439 TG method. Triplicate tissues were treated with the test item for an exposure period of 15 minutes.  The test item was found to have the potential to cause color interference with the MTT endpoint therefore additional tissues were incorporated into the testing to correct for this.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours.  At the end of the post exposure incubation period each tissue was taken for MTT-assay. DPBS was used as negative control and SDS5% w/v was used as positive control. Optical density was measured at 570nm to determine cellular viability. The relative mean viability of the test item treated tissues was 79.7% after the 15 Minute exposure period and 42 Hours post exposure incubation period.

Justification for classification or non-classification

According to the results of the availables key studies, when tested in in vitro assays (ICE assay : Prinsen, 2016, Klimisch 1, GLP, OECD 439) (HCM Test : Duschl, 2007, GLP, Klimisch 1, OECD 492), the test item showed occular irritation. When tested on human cornea model, an irritant potential was shown, however no classification can be made. When tested in ICE Assay, the test item showed irritancy but not sufficient to be considered as Category 1 as Corrosive. With histopathological analysis, the test item showed erosion of the epithelium in this test system. Hence, the test item was classified as Category 2A H319 "causes serioux eye irritation" for Eye Irritation according to CLP criteria.

According to the key studies (Warren, 2018, Klimisch 1, GLP, OECD TG 431 & 439), the test article HC Yellow No. 2 did not induced irritation or corrosion when applied in in vitro dermal test sytem. Hence it was not classified for skin irritation according to CLP criteria.