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Genotoxicity data

Mutagenicity in bacteria (Ames test)

a) First test (Report no. 239005, CCR, D-Rossdorf, July 30, 1991)

This assay was performed according to OECD guideline 471 using S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and TA1538. Incubations were performed with and without metabolic activation. The test substance with a purity of approximately 91% was used. A dose-dependent increase in the mutant frequency was observed in the strains TA98, TA1537 and TA1538. The effect was less pronounced in the presence of the metabolic system. No toxic effects were observed up to the highest dose of 5 mg per plate.

 

b) Second test (Report no. 800463, Ciba-Geigy Ltd, CH-Basle, July 2, 1980)

In this assay S. typhimurium TA98, TA100, TA1535 and TA1537 and TA1538 were incubated with the test item (approx. 95% act. ingr.) with and without metabolic activation. A dose-dependent increase in the mutant frequency was observed in the strains TA98, TA1537 and TA1538. The effect was less pronounced in the presence of the metabolic system.

 

Both tests show that the test substance in two different batches induces frameshift mutations and that the mutagenic component is less mutagenic in the presence of a metabolic system.

Experimental data on putative by-products or degradation products (Ames test) 

a) Ames test with methoxyphenylenediamine dihydrochloride (Report no. 800466, Ciba-Geigy Ltd, CH-Basel, January 20, 1982)  

Without metabolic activation, methoxyphenylenediamine was toxic in strains TA100 and TA1535 and caused an increase in mutant frequency in TA 98 and TA1537. In the presence of the metabolic system, mutagenicity was observed in TA98, TA100 and TA1537.  

 

b) Ames test with 2,4-diaminophenol dihydrochloride (Report no. 800465, Ciba-Geigy Ltd, CH-Basel, January 20, 1982)  

Without metabolic activation, 2,4-diaminophenol dihydrochloride was toxic and caused an increase in mutant frequency in TA 100 and TA1537. In the presence of the metabolic system, mutagenicity was observed in TA98, TA100 and TA1537.  

 

c) Ames test with bis(2-methoxy-3-aminophenyl-)oxadiaziridine (Report no. 80/0464, Ciba-Geigy Ltd, CH-Basel, August 11, 1980)  

Without metabolic activation, the azoxy compound was weakly mutagenic in TA98. The effect was more pronounced in the presence of the metabolic system and occurred in strains TA98 and TA1537.

 

Mutagenicity in mammalian cells in vitro

a) HPRT-test in V79 cells (Report no. 259705, CCR, D-Rossdorf, February 18, 1992)

This assay was performed according to OECD guideline 476. Incubations were performed with and without metabolic activation. The test substance with a purity of approximately 91% was used. The plating efficiency was reduced at concentrations of 0.2 mg/mL without metabolic activation and 0.5 mg/mL with metabolic activation. Doses of up to 1 mg/mL and 1.5 mg/mL were evaluated. There was no dose-dependent increase in the mutant frequency.

 

b) Mouse-lymphoma-assay in vitro (Report no. 801303, Ciba-Geigy Ltd, CH-Basle, February 4, 1981)

This test is based on the method published by Fischer, G. A., Lee, S. Y. and Calabresi, P. in Mutation Researchin 1974.

One million L5178Y cells were incubated with the test substance (approx.. 90-95% act. ingr.) under conditions that kill 80% of the cells, specifically 1.4 mg/mL for 4h and 0.716 mg/mL for 18h. This test was performed without a metabolic activation system. Cultures were allowed to recover for three days and then selected for mutants with methotrexate, cytosine arabinoside or thymidine. There was no increase in mutant frequency.

 

Mutagenicity in lymphoma cells in vivo

Mouse-lymphoma-assay (host-mediated) (Report no. 801301, Ciba-Geigy Ltd, CH-Basle, February 23, 1981)

Groups of four mice were inoculated intraperitoneally with 106L5178Y cells. Three days later, the animals were treated with a single dose of 530 mg/kg bw of the test substance (approx. 90-95%) or vehicle only. Three days after treatment, the animals were sacrificed, the L5178 cells recovered and selected for mutants with methotrexate, cytosine arabinoside or thymidine. Since the intact animal possesses the ability to detoxify and eliminate the potentially mutagenic compound, or alternatively the compound may only produce mutagenic effects after metabolic activation, the host-mediated assay is conducted to allow inferences regarding the effects of the compound in vivo. There was no increase in mutant frequency.

 

Genotoxicity in bone marrow cells in vivo

Nucleus anomaly test in Syrian hamsters (Report no. 801302, Ciba-Geigy Ltd, CH-Basel, January 20, 1982)

For the nucleus anomaly test in somatic interphase nuclei, Syrian hamsters were given daily doses of 360, 720 or 1440 mg/kg bw for two consecutive days. The animals were sacrificed after the second application. Groups consisted of six female and six male hamsters. Bone marrow smears were prepared on slides. The slides of each three male and three female animals were evaluated. For this, 1000 bone marrow cells were scored for single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts or leucopoietic cells and polyploidy cells. Control animals were treated with the vehicle or cyclophosphamide. In contrast to cyclophosphamide, the test substance (approx. 90-95% act. ingr.) did not cause an increase in anomalies in bone marrow cells. There is no documentation of systemic toxicity. In the separate acute oral toxicity study with Syrian hamsters, the LD50 was determined to be 4318 mg/kg bw. At necropsy, no gross organ changes were observed. In mice, the LD50 was determined to be 1592 mg/kg bw, and there were no gross organ changes upon necropsy.

From that it is deduced that oral exposure of Syrian hamsters to TK 11 181 results in systemic availability.

   

Conclusion

The test substance induced frameshift mutations in the absence of a metabolic system in two Ames tests performed with different batches (91% act. ingr. as well as 95% act. ingr.). Mutagenicity was reduced, but not abolished by the presence of rat liver homogenate supplemented with cosubstrates. Considering the size, charge and stability of the azo dye and the fact that the increase in mutant frequency occurred at rather high concentrations, it is suspected that the mutagenicity is caused by a minor component rather than the azo dye itself. Three putative by- or degradation products were all determined to be mutagenic in separate experiments. They gave positive results preferably in strains responsive to frameshift mutagens both in the presence and absence of a metabolic system.

 

The test substance (90-95% act. ingr.) was cytotoxic, but not mutagenic to mammalian cells in vitro. There are no data availble on clastogenicity in vitro. A nucleus anomaly test did not show any genotoxic effect in vivo. With the exception of the hprt test, the data with mammalian cells was obtained using non-standard protocols.

 

There are three possible explanations for the mutagenicity observed in the Ames test:

a) The mutagenicity observed in the Ames test is caused by an impurity. The impurity is a direct acting mutagen. It would not be detected by the micronucleus test or the hprt because these tests are less sensitive than the Ames test.

b) The bacterial Ames test is false positive because the main component may only enter the tester strains because of the rfa mutation which results in a “leaky" cell wall. There is no concern for mutagenicity in mammalian cells.

c) The impurity is a direct acting mutagen in bacteria. It is however sufficiently detoxified in mammalian cells.There is no concern for genotoxicity in humans.

 

In conclusion, the test substance is considered to be not genotoxic.

 


Justification for selection of genetic toxicity endpoint
GLP-conform study according to OECD guideline in mammalian cells

Short description of key information:
The test substance induced frameshift mutations in the absence of a metabolic system in two Ames tests performed with different batches (90-95% act. ingr.). Three putative by- or degradation products were all determined to be mutagenic in seperate experiments. However, the test substance (90-95 act. ingr.) was cytotoxic, but not mutagenic to mammalian cells in vitro. An nucleus anomaly test in vivo did not show any genotoxic effect. A mouse lymphoma asseay (host-mediated) which is conducted to allow interferences regarding the effects of the compound in vivo indicated no increase in mutant frequency. In conclusion, the test substance is considered to be not mutagen.

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 genotoxic under Regulation (EC) No 1272/2008,as amended for the sixth time in Directive EC 605/2014.

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