Disodium (Z)-4-(9-octadecenylamino)-4-oxo-2(or 3)-sulphonatobutyrate

Endpoint summary

Description of key information

Evaluation was based on read-across with subgroup members. Read-across substance 'Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts' containing 25.5% active ingredient was predicted to be non-corrosive and non-irritant to human skin based on the three-dimensional EST 1000 human skin model. Viability values were 96.7% and 102.9%  in the corrosivity test and 126.4% in the irritation test (all >50% threshold values),  therefore it can be considered to be non-irritant to human skin and no further testing is needed according to ECHA progress report 2010.The same substance was predicted to be irritant by means of the in vitro EpiOcular Eye Irritation Test (EIT): relative tissue viability was  39.7% compared to control, which was below 60% cut-off value.  in a BCOP test, test item showed an IVIS score of 15.4 which did not reach the cut-off value of 55.1 and, hence the test item was consequently not predicted to induce severe eye damage or to be corrosive for the eye.  There was also an in vivo eye irritation study in rabbits with read-across substance 'Butanoic acid, 4-amino-4-oxosulfo-, N-tallow alkyl derivs., disodium salts', showing copious lacrimation shortly after application, a moderate degree of conjunctivitis and edema of the lids within four hours and slight opalescence of the cornea in one case. The erythema and edema subsided during the 24 hours,however no scores were available. A subgroup CLP1 classification for eye irritation was concluded, wiht concentration limit of 25.5% for CLP2 classification. 

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 (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Assessment of skin irritation was based on a weight-of-evidence approach:

No data were available for registered substance, however data were available for read-across substance 'Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts'. Justification for read-across with category members is provided in Section 13.

- In a first in vitro study, the corrosive properties of the registered substance were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item containing 25.5% active ingredient was applied for exposure times of 3 minutes or 1 hour. The mean viability versus controls of cells exposed to the test item was 96.7% after a 3-minute exposure period and 102.9% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this human skin model and is predicted to be non-corrosive to human skin.

- In a second in vitro study, the irritant properties of the registered substance to skin cells were also studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013b). The test item containing 25.5% active ingredient was applied for an exposure time of 20 minutes, followed by refreshment of the medium and further 42 hours incubation. The mean viability of cells exposed to the test item was 126.4% of the negative controls and, hence, well above the 50% cut-off value. The test item was considered to be non-cytotoxic and is predicted to be not irritant to skin.

- According to ECHA progress report 2010 (p. 32), it is accepted that in vitro methods for skin irritation represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. A negative result in the human skin model for irritation does not need to be confirmed by additional testing. In conclusion, the read-across substance was predicted to be non-corrosive and non-irritant to human skin.

 

Assessment of eye irritation was also based on a weight-of-evidence approach:

No data were available for registered substance, however data were available for read-across substances

- 'Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts'

- CAS No. 68988-69-2 'Butanoic acid, 4-amino-4-oxosulfo-, N-tallow alkyl derivs., disodium salts'.

Justification for read-across with category members is provided in Section 13.

- In a first in vitro study with 'Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts', test item containing 25.5% active ingredient was tested by means of the BCOP test method (Leuschner, 2013).Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 2.55-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. An IVIS score of 15.4 was calculated, hence the test item was not classified as a severe irritant and not corrosive, based on the results of this test. The corneas treated with the positive control item 1% NaOH solution revealed an IVIS score of 83.5 which was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. The test item, containing 25.5% active ingredient was consequently not predicted to induce severe eye damage or to be corrosive for the eye.

- In a second in vitro study with 'Butanoic acid, 4-amino-4-oxo-2(or 3)-sulfo-, N-(C16-C18 (even numbered), C18 unsaturated alkyl), disodium salts', the eye irritancy potential of test item containing 25.5% active ingredientwas tested by means of the EpiOcular Eye Irritation Test (EIT) (Flügge, 2013c). This model predicts the acute eye irritation potential to stratified human keratinocytes in a three-dimensional structure. Relative tissue viability is determined against the negative control-treated constructs by the reduction of the vital dye MTT compared to negative control. 50 µL of test item, the concurrent negative control or positive control administered for 30 minutes and incubated for 180 minutes with MTT showed a mean cell viability of 39.7% compared to control, which was below threshold of < 60%. The test item was considered to be cytotoxic and predicted to be irritant. The viability of cells treated with the positive control 0.3% Triton®-X-100 was 42.6% of the negative controls and also below the 60% cut-off value. The test item, containing 25.5% active ingredient was consequently predicted to induce severe eye irritation.

- In addition, there was an in vivo eye irritation study in rabbits with read-across substance CAS No; 68988-69-2 'Butanoic acid, 4-amino-4-oxosulfo-, N-tallow alkyl derivs., disodium salts' (Shaffer and Golz, 1956a). Approximately 10 mg of the test item containing 35% active ingredient was instilled into the conjunctival sac of the right eye of each of five albino rabbits. The lids were held closed for about 30 seconds following introduction of the dose, and the eye was examined immediately thereafter and again at intervals over the next several days. There was copious lacrimation shortly after application of the dose, and a moderate degree of conjunctivitis and edema of the lids developed within four hours. A slight opalescence of the cornea was present in one case. The erythema and edema subsided during the 24 hours following the dose, and was not observed after this time. Lacrimation continued to be evident in the case of the animal noted. From the third day till the end of a total 7-day observation period, all eyes maintained a normal appearance. There was no scoring for this study, therefore it was not considered a key study but weight-of-evidence.

- According to Column 2 of the REACH legislation Annex VIII, the criteria for classification are met as irritating to eyes, based on weight-of -evidence. The approach followed was a top-down approach based on in vitro testing as described by Scott et al., 2010 (publication attached) confirmed by weight-of-evidence from in vivo testing. 

-In conclusion, the read-across substance was predicted to induce severe eye irritation, however a subgroup CLP Category 12 classification for eye damage was proposed for the this subgroup.

Conclusion

- In conclusion, the test substance was predicted to be non-corrosive and non-irritant to human skin based on the three-dimensional EST 1000 human skin model. However a subgroup CLP Category 2 classification is given, with limit concentration of 25.5% for non-classification.

- For the eye, the test substance was predicted to be non-corrosive but irritant based on the EpiOcular Eye Irritation Test (EIT) model, confirmed by in vivo testing. A subgroup Category 1 classification is proposed, with limit concentration of 25.5% for Categroy 2 classification.

- This is also supported by other data in the N1 subgroup for structurally similar substances. More information on the subgroup classification is provided in the read-across justification, separately attached in Section 13.

Justification for selection of skin irritation / corrosion endpoint:
Although the in vitro study for irritation was selected, the corrosion study was equally valuable in a weight-of-evidence approach.

Justification for selection of eye irritation endpoint:
The in vitro EpiOcular study predicted irritation, therefore it was selected, allthough the others were equally important in a weight-of-evidence approach.

Effects on eye irritation: irritating

Justification for classification or non-classification

The substance was not skin irritating, however according to CLP regulation (No. 1272/2008 of 16 December 2008) a subgroup CLP Category 2 classification is given, with signal word 'warning' and hazard statement: H315-Causes skin irritation, with concentration limit of 25.5% for non-classification.

The substance is irritating to eyes. According to CLP regulation (No. 1272/2008 of 16 December 2008), a subgroup for Category 1 classification is given, with signal word 'danger' and hazard statement: H318-Causes serious eye damage. However a concentration limit of 25.5% for CLP classification 2 with signal word 'warning' and hazard statement: H3195-Causes seeirous eye irritation.