Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with 4-[2-(4-aminophenyl)diazenyl]-3-methylbenzenamine and methanol sodium salt (1:1)

Endpoint summary

Additional information

The test item was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e.

Salmonella typhimurium and Escherichia coli, in a reverse mutation assay including prival modification at concentrations of 33 μg - 5 000 μg/plate (SPT) and 33 μg - 5 000 μg/plate (Prival) in presence and absence of a metabolic activation system (liver S9 mix from induced rats and uninduced hamster). Precipitation of the test substance was found from about 33 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1 000 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test either without S9 mix or after the addition of a metabolizing system.

In a second non-prival Ames test the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (strain TA 98, TA 100, TA 1535 and TA 1537). The investigations were performed with the following concentrations of the test substance: 25, 75, 225, 675 and 2025 µg/0.1 mL, with and without microsomal activation (Aroclor 1254 induced rat liver S9 mix). At the following concentrations 75, 225, 675, 2025 µg/0.1 mL, with and without metabolic activation, the substance precipitated in soft agar. No evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Read across justification

Toxicity after repeated dose administration of the test item was not evaluated; reliable, experimental data of an analogue are available.

The substances share high similaritiy in structure and have comparable physico-chemical properties. Both substances are solids of poor water solubility and insoluble in most of the common organic solvents. The molecular weight of both compounds is higher than 600 g/mol. The molecules includes phthalimid-like structures and bear the potential to release chlorinated phthalimid after enzymatic or bacterial cleavage. Therefore, the analogue substance was choosen to examine genotoxicity.

At first, the analogue was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. No relevant inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

In the course of a second study, the analogue compound was assessed for its potential to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in hepatocytes of Wistar rats in vivo at 3-hour and 14-hour sampling time. For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were anesthetized and the hepatocytes were harvested by in situ liver perfusion 3 and 14 hours after administration of the test substance. After an attachment period of at least 2 hours the cells were incubated for 4 hours with radiolabeled thymidine in vitro. After washing the hepatocytes were cultivated overnight until fixation. After autoradiography and hematoxylin-eosin-staining three animals per test group with at least 100 cells per animal were scored for DNA repair activity (incorporation of radiolabeled thymidine). No signs of toxicity were observed after administration of 1 000 mg/kg and 2 000 mg/kg body weight at both sacrifice intervals. No reduced viability of hepatocytes as indication for test substance induced toxicity was observed. The single oral administration of the test item did not lead to an increase in the mean net nuclear grain counts at any dose level at both sacrifice intervals.

Short description of key information:
The test item was evaluated in two Ames tests as well as in an UDS and a micronucleus assay in vivo (according GLP and concurrent OECD guideline). No mutagenicity was observed in the standard plate test or in the prival preincubation test in the absence and the presence of metabolic activatio. The substance did not induce unscheduled DNA synthesis or micronuclei in vio.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.