4-(phenylazo)benzene-1,3-diamine

Endpoint summary

Description of key information

Short term toxicity to fish:

A 96 h study was conducted for the substance 4-(phenylazo)benzene-1,3-diamine CAS No. 495-54-5 on Zebra fish(Danio rerio), at a test concentrations of 0.0625mg/L, 0.125mg/L, 0.25mg/L, 0.5mg/L, 1mg/L . The fishes were moving slowly as compared to control. No mortalities were found in the control aquaria. The lethal concentrations LC50 for 4-(phenylazo)benzene-1,3-diamine was found to be >1 mg/L.

Short term toxicity to aquatic invertebrates:

The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 mg/L on the basis of mobiity inhibition effects in a 48 hour study.

Toxicity to aquatic algae and cyanobacteria:

The median effective concentration (EC50) for the test substance, 4-(phenylazo)benzene-1,3-diamine, in Desmodesmus subspicatus was determined to be 0.17 mg/L on the basis of effects on growth rate in a 72 hour study.

Toxicity to microorganisms:

50 % growth inhibition concentration (IGC50) of the test substance 4-(phenylazo)benzene-1,3-diamine on mixed culture of microorganisms was observed to be 0.087 m.mole/L. The study concludes that the test substance showed strong growth inhibition effect on the microorganisms.

Additional information

Short term toxicity to fish:

Different experimental studies of the substance4-(phenylazo)benzene-1,3-diaminewere reviewed for Short term toxicity to fish endpointand are summarised as follows:

Fish Acute Toxicity test according to OECD Guideline 203 was conducted for 4-(phenylazo)benzene-1,3 -diamine The nominal concentration selected for the experiment were 0.0625mg/L,0.125mg/L,0.25mg/L,0.5mg/L,1mg/Land test fish were exposed to these concentration for 96 hours. The lethal concentrations LC50 was found to be >1 mg/L.

LC0 (96 hours) (highest loading at which no mortality was observed) = 1 mg/L

LC50 (96 hours) Experimental = >1 mg/L

LC100 (96 hours) (lowest loading at which 100% mortality was observed) = No mortality was observed.

From peer reviewed journal (Yasuhide Tonogai et al;The Journal of Toxicological Sciences,1982) Short term toxicity to fish was performed for the test material 2,4 Diaminoazobenzene using test organism Oryzias latipes.The test material was dissolved in 1 mL of ethanol and then diluted with water at a concentration of 0.5 mg/L (500 µg/L)and 0.3 mg/L (300 µg/L) for the 24hrs and 48 hrs study period respectively. The LC50 for the test material 2,4 Diaminoazobenzene is 0.5 mg/L for 24 hrs duration and 0.3 mg/L for 48 hrs duration.

Considering the above data, the LC50 value of the substance for fish is observed to be 0.3 - >1 mg/L. This result is not conclusive for classiying a chemical, but considering the harmonised classification of the substance and for the purpose of classification and chemical safety assessment the substance4-(phenylazo)benzene-1,3 -diamine is considered to classified as Aquatic acute 1 and aquatic chronic 1 category.

Short term toxicity to aquatic invertebrates:

Different studies, experimental and predicted, of the substance4-(phenylazo)benzene-1,3-diamine were reviewed for Short term toxicity to aquatic invertebrate

endpoint and are summarised as follows:

Determination of the inhibition of the mobility of daphnids was carried out with the substance4-(phenylazo)benzene-1,3-diamineaccording to OECD Guideline 202. The test substance was tested at the concentrations0, 0.06, 0.12, 0.25, 0.50 and 1 mg/L. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Daphnia magna was determined to be 0.42 mg/L for immobilisation effects.

Supporting above data, Range finding study to Daphnia magna was carried out for 24 hrs. in scientific journal (Aquatic Toxicology, 2011). Neonates (3rd brood) were taken for the experiment. The test neonates were of <24h old. The test chemical conc. used for the study were 0.0248, 0.1243, 0.248, 0.497 and 0.746 mg/l, respectively (0.1, 0.5, 1, 2 and 3 µM). The Chlorella vulgaris was used to feed the D. magna (derived from a stock cultured at the Culture Collection of Algae and Protozoa (CCAP) Argyll, Scotland)were maintained in Bold’s Basal Medium; 0.431 mM K2HPO4, 1.29 mM KH2PO4, 0.304 mM MgSO4·7H2O, 2.94 mM NaNO3, 0.17 mM CaCl2·2H2O, 0.428 mM NaCl, 0.428 mM EDTA-Na4, 1.38 mM KOH, 44.8µM FeSO4·7H2O, H2SO4, 0.462 mM H3BO3,4.9µM ZnSO4·7H2O, 1.17µM MnCl2·4H2O, 1.1µM CuSO4·5H2O,0.275µM Co(NO3)2·6H2O, 0.79µM Na2MoO4·2H2O in a fermentervessel at 20±1◦C, under a 16-h:8-h light : dark cycle with light providedby a Gro-Lux photosynthetic tube.To provide a food stock, cells were collected by centrifugation(2250×g, 30 min, 20◦C) and re-suspended in a volume of waterto give an OD (1:10) of 0.8 at 440 nm. Groups of 10 neonates (<24 h old) in 100 ml fresh media were exposed to increasing concentrations of test chemical for 24 h (n= 2 test groups per concentration). After 24 h, any mortality and abnormal behaviour or appearance was recorded. The test was performed under the following conditions- pH-6 - 8 pH, Hardness-140 – 250 mg/l as CaCO3,Temp.-20 ± 1°C ,Conductivity-360–480 µS cm⁻¹.Viability was analyzed by the number alive and speed of movement as compared to the control cultures. Based on percentage viability of test organism Daphnia magna, the LC0 and LOEC value was found to be 0.497 and 0.746 mg/l, respectively.

Further supporting data was based on the QSAR prediction done using the EPI Suite ECOSAR version 1.11 in which the 48 hours EC50 was estimated to be 0.778 mg/L on Daphnia Magna for substance 4-(Phenylazo)benzene-1,3-diamine with immobilization effects.

Thus considering the key and supporting values, it can be concluded that the substance is likely to be hazardous to aquatic invertebrates and can be classified as Aquatic acute category 1 as per the CLP criteria. But considering the harmonised classification of the substance and for the purpose of classification and chemical safety assessment the substance4-(phenylazo)benzene-1,3 -diamine is considered to classified as Aquatic acute 1 and aquatic chronic 1 category.

Toxicity to aquatic algae and cyanobacteria:

Different studies, experimental and predicted, of the substance 4-(phenylazo)benzene-1,3-diamine were reviewed for Short term toxicity to aquatic algae and

cyanobacteria endpoint and are summarised as follows:

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance4-(phenylazo)benzene-1,3-diamineaccording to OECD Guideline 201. The test substance was dissolved in OECD growth medium and tested at the concentrations0, 0.5, 0.10, 0.20, 0.40 and 0.80 mg/L. Effects on the growth rate of the organism were studied. The median effective concentration (EC50) for the test substance,4-(phenylazo)benzene-1,3-diamine, in Desmodesmus subspicatus was determined to be 0.17 mg/L.

Supporting data from other lab indicates, the effect of test item 4-(phenylazo)benzene-1,3-diamine, CAS No. 495-54-5 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.9 mg/L,2.7 mg/L,8.1 mg/L,24.3 mg/L,72.9 mg/L,218.7 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 10.07mg/L.

Further supporting data is based on the QSAR prediction done using the EPI Suite ECOSAR version 1.11, the 72 hours EC50 was estimated to be 0.653 mg/L on algae

for substance 4-(Phenylazo)benzene-1,3-diamine with growth inhibition effects.

Thus considering the key and supporting values, the EC50 value is observed to be in the range 0.17 - 10.07 mg/L. But considering the harmonised classification of the substance and for the purpose of classification and chemical safety assessment the substance4-(phenylazo)benzene-1,3 -diamine is considered to classified as Aquatic acute 1 and aquatic chronic 1 category.

Toxicity to microorganisms:

In order to evaluate the effect of test substance 4-(phenylazo)benzene-1,3-diamine on the biological waste treatment, the growth inhibition effect of it on activated sludge microorganisms was studied. Growth inhibition effect was evaluated by the concentration of test compound in which logarithmic phase growth rate constant was decreased to 50 % of that of the culture without test compound. Growth rate constant was determined with turbidity of that of the culture at 660 nm. The mixed microorganisms were obtained from supernatant of activated sludge and pre-incubated with the medium without azo compounds for 2 days. The microorganisms were inoculated to the experimental medium to give the optical density of the culture at 0.02-0.05. Incubation temperature was at 25±1 deg C. 50 % growth inhibition

concentration (IGC50) of the test substance 4-(phenylazo)benzene-1,3-diamine on mixed culture of microorganisms was observed to be 0.087 m.mole/L. The study concludes that the test substance showed strong growth inhibition effect on the microorganisms.