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

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Description of key information

STATEMENT ON FISH BIOACCUMULATION STUDY, ENVIGO 2017 (OECD TG 305)

Envigo Study Number:  PP78VH

Black 400: Bioaccumulation in fish

Summary of pre-exposure tank water analysis:

Nominal concentration of [¹⁴C] Black 400 in tank water = 5 µg/L

Diluent water flow rate maintained at 800 mL/minute.

[¹⁴C] Black 400 stock solution prepared in DMF; flow rate into diluent water was maintained at 0.59 ‑ 0.62 mL/hour.

Measured Concentrations of [¹⁴C] Black 400 in tank water (determined by radioassay) were highly variable (1.93 – 8.01 µg/L) and not within the acceptance criteria for validity of the study.

Due to the variability in concentrations of [¹⁴C] Black 400 in tank water, further investigations were conducted which indicated and accumulation of radiolabelled test item at the surface of the water, within the test tank (112 µg/L). Radioactive concentrations at other areas of the test tank, sampled contemporaneously with the surface water sample, fell within the range previously described. Due to the significant accumulation of the test item at the surface of the water in the test tank, it was concluded that the aqueous route was not a viable route of exposure.

The dietary route of exposure was therefore considered as a regulatory acceptable alternative but has been ruled out by Envigo aquatic toxicity experts as not suitable for the PBT assessment of an industrial chemical on the basis that the methods to estimate BCF from measurements done in a feeding study are all methodologically weak.

Additional information

A fish bioconcentration study with aquatic exposure conducted in 1993 did not reach a steady-state of accumulation after 28 days (95% steady state being predicted after 48 days) and it is not possible therefore to classify the test substance as B or vB. It is therefore classified as unassignable Klimisch 4.

A validated QSAR (BCFBAF v3.01) shows that the BCF is  less than 2000 (BCF 5551.7 L/g). The experimental value of Log Pow 4.66 was used in the calculation and this value is supported by Log Pow calculated from the solubility in water and octanol:

Solubility in octanol = 500 mg/l

Solubility in water = 0.02 mg/l

Log Pow by solubility = 500/0.02 = log Pow 4.4

A further bioaccumulation study in fish was undertaken in accordance OECD TG 305, as a result of ECHA Decision on Testing Proposal set out in a registration pursuant to article 40(3) of regulation(EC) NO 1907/2006. Aquatic exposure was attempted but radioactive concentrations in the test tanks were variable, and generally low. After further investigation, it was found that concentrations of the test item were very high at the surface of the water suggesting that a film is forming due to the low aqueous solubility of the test item. The test item has been prepared as a solution in DMF which is combined with diluent water, in a mixing vessel, prior to flowing into the bottom of the test tank at a rate of 80 mL/minute. The outlet of the test tank is at the top of the tank at the opposite end to the inlet to aid mixing within the tank.

Dietary exposure was ruled out. The fish bioaccumulation study with dietary exposure is designed for testing of chemicals with direct oral exposure to humans e.g. pharmaceutical substances and is not intended for the determination of an environmental BCF.  While a result from the dietary study can be converted to a BCF value the conversion algorithm contains so many safety factors it renders the final result meaningless.

In the previous study, the Black 400 was prepared in acetone + 1% Tween 80. Although acceptable according to the guideline, acetone is no longer used as it promotes algae growth and the use of the dispersant means that the test item is not truly bioavailable.