Reactions mass of N-{2-[(4-bromo-6-substituted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-methoxyalkyl)amino]phenyl}acetamide) and N-{2-[(4-bromo-6-substiuted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-hydroxy-3-phenoxypropyl)amino]phenyl}acetamide

Endpoint summary

Additional information

Genetic toxicity in vitro

- Gene mutation in bacteria

This study was performed to investigate the potential of FAT 41044/A TE to induce gene muta­tions according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate; Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred most of the strains. A minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1535 and TA 1537 in experiment I without S9 mix at 2500 µg/plate and in experiment II in strain TA 1537 in the absence of metabolic activation at 5000 µg/plate and in strain TA 1535 in the presence of metabolic activation at 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41044/A TE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Conclusion :In conclusion, FAT 41044/A TE is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

-Chromosome aberration test in mammlian cells

The test item FAT 41044/A TE, suspended (Exp. I) or dissolved (Exp. II) in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:   Without S9 mix With S9 mix   Exp. I Exp. II Exp. I & II Exposure period  4 hrs 22 hrs  4 hrs Recovery 18 hrs - 18 hrs Preparation interval 22 hrs 22 hrs 22 hrs In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (3200.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations and thus concentrations at the border of solubility were chosen for evaluation of cytogenetic damage. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, FAT 41044/A TE is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Cell gene mutation test in vitro

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at ten dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 Experiment 1

Group	Concentration ofFAT41044/A TE(µg/mL) plated for mutant frequency
4-hour without S9	39, 78, 156, 208, 260, 312
4-hour with S9 (2%)	39, 78, 156, 208, 260, 312

 Experiment 2

Group	Concentration ofFAT41044/A TE(µg/mL) plated for mutant frequency
24-hour without S9	39.06, 78.13, 156.25, 312.5, 625, 1250
4-hour with S9 (1%)	78.13, 156.25, 312.5, 625, 1250, 2500

Results: The maximum dose level used in the Mutagenicity Test was limited by a combination of test item-induced toxicity, and the presence of precipitate effectively reducing exposure of the test item to the cells. Overall, precipitate of the test item was observed at and above 4.88 µg/mL in the Mutagenicity Test. The vehicle controls (DMSO) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion: The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Genetic toxicity in vivo

Mammalian erythrocyte micronucleus test

The test item FAT 41044/A TE was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was suspended in DMSO / PEG 400 (3/7), which was also used as vehicle control. The volume administered orally was 10mL/kg b.w. The volume of the positive control administered was 10 mL/kg. The faeces of the animals were blue coloured after treatment with the test item in the pre-experiment and in the main experiment for the mid and high dose group.

24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. At least per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

The maximum guideline-recommended dose of 2000 mg/kg b.w. was investigated at preparation intervals of 24 and 48 h, respectively, based on two pre-experiments.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that FAT 41044/A TE did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle control there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. In fact the mean values of micronuclei observed after treatment with FAT 41044/A TE were below to the value of the vehicle control group.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 41044/A TEdid not induce micronucleias determined by the micronucleus test with bone marrow cells of the mouse.Therefore, FAT 41044/A TE is considered to be non-mutagenic in this micronucleus assay.

Justification for selection of genetic toxicity endpoint
No study was selected, since the available genetic toxicity studies were negative.

Short description of key information:
In vitro:
Negative Ames test (OECD 471) with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia colistrain WP2 uvrA., with and without metabolic activation
Negative result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, with metabolic activation
Positive result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, without metabolic activation
In vivo:
Negative results in an in vivo erythrocyte micronucleus test (OECD 474) in mice, with and without metabolic activation
Negative result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, with and without metabolic activation
Positive result in a chromosome aberration test (OECD 473) using Chinese hamster V79 cells, without metabolic activation
In vivo:
Negative results in an in vivo erythrocyte micronucleus test (OECD 474) in mice

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).