Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

Endpoint summary

Description of key information

The aquatic toxicity potential of substance FAT 40549 was evaluated in studies conducted with fish, invertebrates, plants (Lemna) and bacteria. In 96 hr Rainbow Trout toxicity test (OECD 203) the LC50 and LOEC value was found to be > 100 mg/L, and acute Daphnia toxicity test (OECD 202) available with the 48 hr EC50 and NOEC values reported as 100 mg/L. Also an 21 days semi-static test following the OECD Guideline No. 211 is available, in which the 21-day EC10 amounted to 0.8 mg/l with a 95 % confidence interval from 0.03 to 2.1 mg/l, and 21 d-NOEC was equal to or higher than 0.8 mg/L.In an OECD 201 guideline test 72 hr EC50 of the test substance on the growth of Scenedesmus subspicatus was noted as 89.9 mg/l whereas the EC10 was 2.8 mg/l. The inhibitory effect of FAT 40'549/A on aerobic waste water bacteria of activated sludge EC50 and EC10 (30 minutes) was determined to be >100 mg/L.

In the Daphnia magna reproduction test which was conducted following OECD test guideline 211 in 1998 under GLP conditions, the 21-day NOEC was determined to be at least 0.8 mg/L, and the 21 -day LOEC was determined to be 1.55 mg/L. The following point are to be noted pertaining to the study report.

1. The test was conducted as per guideline and GLP with no deviations which demands the study to be categorized as Klimish 1 and hence as a Key study.

2. In the test media the test sample was observed to degrade into a defined reaction product. In the 48 hours-aged samples the mean measured

concentrations had decreased to 17 - 35 % of the nominal values, and in the 72 hours-aged samples to 4 - 32 % of nominal (quantification based only on the main compound).

3. The survival rate at the end of 21-day exposure is 80% at the highest tested concentration of 20 mg/L.

There is a 1996 acute Daphnia toxicity test (OECD 202) available with the 48 hr EC50 and NOEC values reported as 100 mg/L.

Further, the substance is found to be not bio-degradable based on the following studies:

1. A ready biodegradation test (1996) conducted as per OECD 301F reported. 2.7% biodegradation at the end of 28 day based on O2 consumption.

2. An inherent biodegradation test (1996) conducted as per OECD 302B reported the substance to be non-biodegradable at the end of 28 day based on DOC removal.

However, according to a hydrolysis study conducted in 1996 according to OECD test guideline 111 the vinylation reaction (t_1/2 (25 °C) < 24 hours) of the main product of FAT 40'549/A and the following hydrolysis reaction (t_1/2 (25 °C) = 81 days) of the vinylated main product formed was estimated to be fast at 25 °C and pH 9. The main product of FAT 40'549/A was considered to be stable at pH 4, since the half-life time for the vinylation reaction was estimated to be more than 1 year at 25 °C. At pH 7, a relative slow vinylation t1/2 (25 °C) = 9.8 days) of the main product of FAT 40'549/A was determined at 25 °C and pH 7, but the vinylated main product formed is considered to be stable at this pH

range (t_1/2 (25 °C) > 1 year.)

In the OECD 211 study the test substance Reactive yellow 205 was found degraded into a defined reaction product in the test media. The decrease of the concentrations during the test medium renewal periods of 48 and 72 hours was obviously caused by degradation of the main compound due to hydrolysis. The degradation of test substance was noted in the 48 hr test media sample, and there was no mortality observed during this time (48 hr) at the highest test concentration of 20 mg/l. Hence, the toxicity observed (reproduction rate) could be due to the degraded product or could be a synergic effect of test substance and degraded product. There is no separate tox data for test substance and the degraded product to confirm this. Therefore, based on the analytical evidence that the test substance degraded in the test medium its proposed not to classify this substance based on chronic test data as the observed results could not be corelated only to the test substance (Reactive Yellow 205). Hence the test substance FAT 40549 is classified as Aquatic Chronic Cat 3.

The proposed classification also falls in line with several other Reactive dyes which are classified as Aquatic Chronic Cat 3.

Additional information

Study reports are available for acute tests with fish, invertebrate, algae, micro-organism and Daphnia reproduction test. The results are summarized as below:

The acute toxicity of the test substance FAT 40'549/A to Rainbow trout was determined in a 96-hour static test according to OECD Guideline No. 203 in compliance with Good Laboratory Practice Regulations. A limit test was performed in accordance with the test guidelines to demonstrate that the test substance has no toxic effect on the test fish up to the concentration of nominal 100 mg test substance/L. The analytically determined test concentrations in the test medium of nominal 100 mg/L ranged from 89 % to 94 % of the nominal value (calculated as the average over all measurements). The 96-hour LOEC (lowest concentration with toxic effects) and the 96- hour LC 50 were higher than 100 mg test substance/L.

The acute toxicity of the test substance FAT 40'549/A to Daphnia magna was determined in a 48-hour static test according to OECD Guideline No. 202 in compliance with Good Laboratory Practice Regulations. The nominal concentrations tested were 4.6, 10, 21, 46 and 100 mg test substance/L, and in parallel a control. The analytically determined test concentrations in the test media ranged from 92 % to 96 % of the nominal values (calculated as the average over all measurements per test concentration). The 48-hour LOEC (lowest concentration with toxic effects) and the 48-hour EC 50 were higher than 100 mg test substance/L.

The influence of the test substance FAT 40'549/A on the growth of the green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) was investigated in a 72-hour static test according to OECD Guideline No. 201 and used as the key study. The test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect caused by reduced light intensities in the coloured test solutions. This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance FAT 40'549/A on Pseudokirchneriella subcapitata was caused only due to the indirect effect, the light absorption in the coloured test solutions. Thus, a real toxic effect of the test substance on the growth of Pseudokirchneriella subcapitata can be excluded up to the highest test concentration of nominal 100 mg test substance/l. Where as in another study conducted following OECD 201 guideline test 72 hr EC50 of the test substance on the growth of Scenedesmus subspicatus was noted as 89.9 mg/l whereas the EC10 was 2.8 mg/l.

The inhibitory effect of FAT 40'549/A on aerobic waste water bacteria of activated sludge was investigated in a respiration test. The respiration rate of aerobic waste water bacteria was not inhibited when exposed to the test article concentrations in the range from 3.2 to 100 mg/L. The EC50 and EC10 (30 minutes) was therefore determined to be >100 mg/L.

The influence of the test substance FAT 40'549/A on the reproduction and survival rate of Daphnia magna was investigated in a 21 days semi-static test following the OECD Guideline No. 211. The 21-day EC10 and EC50-values for the reproduction rate of the test animals were calculated by Probit analysis (based on mean test substance concentrations). The 21-day EC10 amounted to 0.8 mg/l with a 95 % confidence interval from 0.03 to 2.1 mg/l, and 21 d-NOEC was equal to or higher than 0.8 mg/L.