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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test (OECD TG 471)


The substance is negative in the L5178Y TK +/- Mouse Lymphoma Assay (OECD TG 490)


The substance is negative in the Chromosome Aberration test (OECD TG 473)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance is negative in the micronucleus cytogenetic assay (OECD TG 474)

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames-Genetic toxicity in vitro, OECD TG 471, RIFM, 1994


Test material (>99% pure) in acetone was tested in the Ames test (OECD TG 471, RIFM, 1994) in accordance with GLP in absence or presence of Aroclor-induced rat liver S9 at doses 0, 10, 33, 100, 333, 1000 and 5000 mg/plate using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia Coli strain WP2 UVRA and appropriate positive controls in triplicate. The doses were based on a preliminary assay. Results: Slight precipitation was seen at the three highest doses (≥333 µg/plate) but no cytotoxicity was not observed. All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed at any dose with any of the six strains with or without activation. Based on this the substance is not mutagenic in this test.


Genetic toxicity in vitro, Chromosomal aberrations, OECD TG 473, RIFM 1995


The substance (purity >99%) is tested in a cytogenetic assay with Chinese Hamster ovary cells (CHO-K1) in the OECD TG 473 and in accordance with GLP (RIFM, 1995). Concentrations of 5, 10 and 20 μg test item/ml were used without metabolic activation, using 4/20, 20/20 and 44/44 hr exposure/harvest periods. In the study with metabolic activation (S9 from rat liver induced by Aroclor 1254), dose levels of 8.7, 17.3 and 34.5 μg HHCB/ml were tested for the 4-hr period with a 20-hr harvest time and dose levels of 22.6, 28.2 and 30.0 μg/ml for the 4-hr period with a 44-hr harvest time. At the 20 and 44-hr harvest times, the cells were assessed for structural chromosome aberrations, and at the 44-hr harvest time, also for numerical chromosome aberrations. Results: The mitotic index was significantly lowered at the highest dose in all cases. N-methyl-N’-nitro-N-nitrosoguanidine was used as a positive control in the non-activated study and benzo(a)pyrene in the activated study. Positive controls (N-methyl-N’-nitro-N-nitrosoguanidine without S9 and benzo(a)pyrene with S9) were included and caused increases in structural (significantly) aberrations in all cases. Cytotoxicity was observed at 20 μg/ml after 20 and 44 hours of treatment without activation and at 30 and 34.5 μg/ml after 4 hours of activation. No significant increase in structural or numerical chromosome aberrations was observed with or without activation with the test item at any dose. HHCB was concluded to be negative for chromosome aberrations in this test. Based on this the substance is not cytogenetic in this test.


Genetic toxicity in vitro - MLA, OECD TG 490, IFF, 2020 


A study was conducted in order to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD TG 490, GLP). One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 9 dose levels (without S9: 0,1,2,4,8,16,20,24,28,32μg/mL and with S9: 0,2,4,8,16,32,48,56,64,72μg/mL) in duplicate, together with solvent (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used was limited by test item-induced toxicity, the 32 μg/mL (+S9) and 64 and 72 μg/mL (-S9) doses were not plated. Results: A precipitate of test item was not observed in the main test. The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances methyl methane sulfonate and cyclophosphamide induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.


Genetic toxicity in vivo - in vivo micronucleus test, OECD TG 474, RIFM, 1997


In the micronucleus test (OECD TG 474, GLP), groups of 5 male (28.1-37.2 g) and 5 female ICR mice (24.5-31.0 g) were dosed with 0, 376, 750, or 1500 mg/kg bw test item (in corn oil - purity >99%) by intraperitoneal injection at a constant volume of 20 ml/kg bw. The high dose was selected to be approximately 70% of the estimated intraperitoneal LD50 based on a preliminary test. The positive control was cyclophosphamide. Bone marrow was harvested at 24, 48 and 72 hr after dosing and examined for micronucleated polychromatic erythrocytes (PCE). No mortality was seen. Lethargy was observed in all animals on 1500 mg/kg bw, in 4/15 males and 4/15 females on 750 mg/kg, and 1/15 males and 0/15 females on 376 mg/kg bw. Moderate reductions (up to 25%) in the ratio of PCE to total erythrocytes were observed in groups on 1500 mg/kg bw after 48 and 72 hrs indicating toxicity and bioavailability to the bone marrow target. The positive control induced a significant increase in micronucleated PCE in both male and female mice at 24 hr (the only harvest time for this group). No significant increase in micronucleated PCE in HHCB-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hr after dose administration. Based on this the substance is not cytogenetic in this test.


 


An overview of the available in vitro and in vivo mutagenicity studies is presented in the table below.




















































































































Type



Species



Activation



Doses



Results



GLP



OECD TG



Reference



In vitro Bacterial Reverse Mutation Assay



Salmonella typhimurium (TA98, 100, 1535, 1537, 1538) & E. coli (WP2 UVRA)



With and without


S9



10, 33, 100, 333, 1000, 3333, 5000 µg/plate



Negative



Yes



471



Api and San, 1999, San and Sly, 1994



In vitro Bacterial Reverse Mutation Assay



Salmonella typhimurium (TA97, 98, 100 and 102)



With and without


S9



5, 16.6, 50, 166.6, or 500 µg/plate



Negative



No



 



Mersch-Sunderman, 1998a



In vitro SOS induction



E. coli PQ37



With and without


S9



0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50µg/assay



Negative



No



 



Mersch-Sunderman, 1998b



In vitro cytogenetic assay



Chinese hamster ovary cells



With and without


S9



9, 17, 34µg/ml & 23, 28, 30 µg/ml



Negative



Yes



473



Api and San, 1999a, Curry and Putman, 1995



In vitro micronucleus test



Human lymphocytes



 



0.05, 0.49, 4.85, 48.5, 97, 194µM (=0.013, 0.13, 1.25, 12.5, 25, 50 µg/ml)



Negative



No



 



Kevekordes et al., 1997



In vitro micronucleus test



Human hepatoma cells



None added, some inherent



0.1, 0.97, 9.7, 97, 194, 387 µM (=0.036, 0.25, 2.5, 25, 50, 100 µg/ml)



Negative



No



 



Kevekordes et al., 1997



In vitro sister chromatid exchange test



Human lymphocytes



With and without


S9



0.025, 0.25, 2.43, 24.25, 48.5, 97µM (= 0.007, 0.07, 0.6, 6, 12.5, 25 µg/ml)



Negative



No



 



Kevekordes et al., 1998



In vitro unscheduled DNA synthesis



Primary rat hepatocytes



Inherent



0.15, 0.5, 1.5, 5, 15, 50µg/ml



Negative



Yes



482



Api and San, 1999, San and Sly, 1994



In vivo micronucleus test



Mice



 



380, 750, 1500 mg/kg bw



Negative



Yes



474



Api and San, 1999, Gudi and Ritter, 1997



DNA precipitation assay and


Micronucleus test



Zebra mussel(Dreissena polymorpha)



 



100 ng/L, 500 ng/L



Significant increase in DNA fragmentation


No significant increases in MN frequency



 



 



Parolini et al., 2015



 

Justification for classification or non-classification

Based on the results of the gene mutation in bacterial cells (Ames test), in vitro cytogenicity (chromosome aberration), in vitro gene mutation in mammalian cells, and the in vivo cytogenicity test, the substance is not genotoxic and therefore does not have to be classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).

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