6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Endpoint summary

Description of key information

Based on available data today, unable to conclude P or B under PBT classification.

Additional information

A review of some of the available information for Black 400 indicates that:

 

it is highly insoluble

it has a high Log Kow, experimental value reported at Log Kow of 4.66

 

Previous ecotoxicity studies generated include:

Ready biodegradability (OECD 301D), HLS1989

·        Test substance was dissolved in chloroform with aliquots placed on Whatman paper, solvent allowed to evaporate, and one piece of paper was introduced into the test medium.

·        Test substance gave 5% biodegradation compared to 89% biodegradation in sodium benzoate control within 28 days, therefore not readily biodegradable

 

Inherent biodegradability (OECD 301B)- Modified STURM test, HLS 1992

·        Test substance was dissolved in chloroform with aliquots placed on Whatman paper, solvent allowed to evaporate, and one piece of paper was introduced into the test medium.This was necessary due to the very low solubility of test substance in water.

·        Pre-treatment of test substance for 6 days to facilitate degradation of background dissolved organic carbon (DOC) to reduce carryover into test vessels.

·        Test substance gave 2% biodegradation compared to 61% biodegradation in sodium benzoate control after 28 days,therefore not inherently biodegradable

 

Overall not readily or inherently biodegradable, which would typically trigger P, vP classification, however in this case, this is most likely due to insolubility of the test substance experienced in the studies rather than the inherent chemical nature of the substance itself.

 

Acute Daphnia (OECD 202), HLS 1989

·        Test substance was prepared in 1% Tween80-THF.

·        Stability of test concentration was verified by chemical analysis at 0 and 48 hours.

·        No mortality occured in 40 daphnids exposed to a test concentration of 1.0mg/L for a period of 48 hours.

·        1.0mg/L was the highest test concentration that could be prepared due to the limited water solubility of the test material in water.

·        No effect levels reported as ≥ 1.0mg/L at 24 and 48 hours.

·        Verification of the test concentrations showed a decline in the test concentrations over a 48hour period to a mean value of 69% of nominal concentration despite attaining near nominal concentrations at 0 hours. This equates to a test concentration of 0.69mg/L, and this should be taken as the true exposure level to give a worst case analysis of the data.

 

Overall not toxic to the daphnia and the aquatic environment, at the concentrations reported

 

Bioaccumulation- A validated QSAR (BCFBAF v3.01) shows that the BCF is  BCF 551.7 L/g.

 

 

Bioaccumulation in Rainbow Trout (OECD 305E), HLS 1993

·        14C study, 19.44uCi/mg, 98% purity

·        Test substance solubility measured as 0.021mg/L in water

·        14C test substance administered to fish continuously as a solution in acetone to provide 2 nominal test concentrations of 5.0 and 0.5ug/L. Due to low water solubility, Tween 80 was added to the acetone stock solutions.

·        Exposure to nominal 0.5ug/L- Mean concentration of radioactivity maintained at 0.55 ± 0.09ug/L. Mean concentrations increased to 0.27 (Day 1), 0.53 (Day 3) and then slowly 2.0 (Day 14) to 2.5 (Day 28) ugequiv/g. BCF increased steadily throughout the study 550 (Day 1) to 4300 (Day 28).

·        Exposure to nominal 5.0ug/L- Mean concentration of radioactivity maintained at 3.5 ± 0.8ug/L. Mean concentrations increased to 2.8 (Day 1), 4.3 (Day 3) and 8.9 (Day 7) then slowly to 15 ugequiv/g at Day 28. BCF increased steadily throughout the study 900 (Day 1) to 4800 (Day 28). Concentrations of total radioactivity were not maintained at the high exposure level and the test substance was only approx. 70% of radioactivity in water at day 28.

·        95% steady state was predicted to be 48 days and a half life of 11 days.

 

Overall steady state of accumulation not reached. At high and low concentrations, BCF reported as 550-900 on Day 1, 3300-3500 on Day 14 and 4300-4800 on Day 28

 

Pre-exposure study for repeat Bioconcentration study, Envigo 2017

·        Nominal concentration of 14C Black 400 = 5ug/L. Stock solution prepared in DMF.

·        Measured concentrations were highly variable 1.93- 8.01ug/L- not within the acceptance criteria for validity of the study

·        Further investigations showed an accumulation of 14C test item at the surface of the water (112ug/L), and decreased concentrations in other areas of the water.

 

Overall, due to significant accumulation of the test item at the surface water, it was concluded that the aqueous route was not a viable route of exposure

 

 

Therefore:

For P

·        Black 400 is not biodegradable, most likely due to insolubility of the test substance experienced in the studies rather than the inherent chemical nature of the substance itself.

·        Remove current voluntary classification as there is no basis for P or vP with current data- i.e. unable to conclude P, vP.

 

For B

·        Existing Huntingdon study- not able to conclude B or vB as steady state of accumulation of test item not achieved.

·        New pre-exposure study conducted has shown that the aqueous rate is not viable/not technically feasible. According to OECD305 Guidance, dietary route of exposure is recommended for bioaccumulation studies of substances with logKow > 6, and water solubility below ~0.01 – 0.1 mg/L. Black 400 does not fit into this criteria- thus unable to conclude B, vB

 

 

Based on available data today, unable to conclude P or B under PBT classification.

 

This is clearly a solubility and possibly a bioavailability issue based on the ecotoxicity studies conducted to date.