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In vitro:


 


In a GLP study performed according to OECD guideline 471, P-#620 was tested for mutagenicity usingSalmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 andEscherichia coliWP2 uvrA-with the preincubation method (20 min) in the presence and absence of metabolic activation system (S-9 mix).


A preliminary test was performed using concentrations between 9.8 and 5000 µg/plate with and without S-9. Due to cytotoxicity, the following concentration ranges were tested in the 2 main tests, using the preincubation method(20 min at 37°C) with and without S-9:


without S-9: 2.4 - 78 µg/plate (strains TA100, TA 1537 and TA1535), 4.9 - 156 µg/plate for TA98 and 313 - 5000 µg/plate forE. colistrain.


with S-9: 4.9 - 156 µg/plate for TA100, 9.8 - 313 µg/plate for strains TA1535 and 1537, 39 - 1250 µg/plate for TA98 and 313 - 5000 µg/plate forE. colistrain. At least five different levels of concentrations were tested in triplicates, for each strain.


The positive controls induced the appropriate responses in the corresponding strains. P-#620 showed no substantial increases in revertant colony numbers over control count obtained with any of the tested strains at any concentrations in either presence or absence of S-9.


Under the test conditions, P-#620 is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EEC.


 


In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473, and in compliance with the GLP, P#620 (purity >99%) diluted in DMSO was tested in female Chinese Hamster lung (CHL) cells in the presence and the absence of mammalian metabolic activation (S9) at concentrations of varying from 22.5 to 700 µg/mL.


P#620 was incubated with the cells for 6, 24 or 48 hours and the cells were analysed for the presence of chromosomal aberrations 18 hours (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period).


Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses.


Cytotoxicity was observed at 700 µg/mL with metabolic activation in the first assay. For the second test, the only maximum dose level (100 µg/mL direct assay, 700 µg/mL for the metabolic activation with S9) of the first test was repeated to test because the negative results were obtained in the first test. No increase in the occurence of chromatid or chromosome aberrations was observed with and without metabolic activation at any tested concentration and for all exposure period tested.


Under the test conditions, P#620 did not show any cytogenic activity in the chromosomal aberrations test using CHL cells according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC.


This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.


 


In a GLP study performed according to OECD guideline 476, The test item AMBER CORE was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Two Main Assays were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 2.00 μL/mL and at a wide range of lower dose levels: 1.00, 0.500, 0.250, 0.125, 0.0625, 0.0313, 0.0156 and 0.00781 μL/mL. In the absence of S9 metabolic activation, no cell survived treatment from 0.0313 μL/mL onwards, while a slight toxic effect was noticed at the next lower dose level (0.0156 μL/mL). In the presence of S9 metabolism, no cell survived treatment at the three highest dose levels. Dose related toxic effects were observed over the remaining concentrations tested, reducing relative survival (RS) to 23% of the concurrent negative control at 0.250 μL/mL.Two Main Assays for mutation to 6-thioguanine resistance were performed. Cells were treated for 3 hours, both in the absence and presence of S9 metabolism and maintained in growth medium for 8 days to allow phenotypic expression of induced mutation. In the first Main Assay, in the absence of S9 metabolic activation, no cell survived treatment at the highest dose level, while at the next lower concentration, relative survival was reduced to 43% of the concurrent negative control. In the presence of S9 metabolism, severe toxicity was observed at the two highest dose levels, while moderate reduction of relative survival (33%) was noticed at the next lower dose level (0.256 μL/mL). In order to evaluate mutagenic effects at adequate levels of cytotoxicity (between 20 and 10% RS), a second assay was performed (Main Assay 2) where more closely spaced concentrations were used. Dose related cytotoxicity was observed in the absence of S9 metabolism reducing relative survival to 23% at the highest dose level. Once again a very steep concentration response curve was observed in the presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment both in the absence and presence of S9 metabolic activation. The mutant frequency of the solvent/vehicle controls fell within the 95% confidence limits of the negative control range. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. Adequate number of cells and concentrations were analysed in bothMain Assays with the exception of the second treatment series in the presence of S9, in which only cultures at the three lowest dose levels survived treatment. No precipitation was noted at any concentration tested in any experiment. No statistically significant or biologically relevant increase in mutant frequency was observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. It is concluded that AMBER CORE does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.


 


 


In vivo:


In anin vivomicronucleus assay performed according to the OECD guideline No. 474, and in compliance with the GLP, Amber core (P#620) (purity unknown) was administratedviaintraperitoneal route to male albino Hsd:ICR (CD-1) mice (7 animals/dose level). In a preliminary test, two mice (one female, one male) were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. In the preliminary test, the test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. No evidence of toxicity was observed in animals dosed with test materialviathe oral route and, therefore systemic absorption could not be confirmed using this dose route. Adequate evidence of test material toxicity was demonstratedviathe intraperitoneal route of administration which was therefore selected in the main test. The maximum recommended dose of the test material, 2000 mg/kg bw, was selected for use in the main test, with 1000 and 500 mg/kg bw as the lower dose levels.


In the main, test, animals were therefore treated with Amber Coreviaone intraperitoneal injection. 24 or 48 Hours after the treatment, animals were killed and bone marrow was collected for further analysis. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated in order to determine the toxicity of the test material.


A modest decrease (but not statistically significant) was observed in the PCE/NCE ratio in both the 24 and 48h test material dose groups when compared to their concurrent control groups. This, together with the observation of clinical signs (Hunched posture, ataxia, lethargy, ptosis and splayed gait) was considered to indicate that systemic absorption occurred and exposure to the target tissue was achieved. Furthermore, there was no statistically significant increase in the incidence of micronucleated PCE.


In conclusion, under the test conditions, Amber Core (P#620) is not considered as genotoxic in thisin vivomicronucleus test. Therefore, Amber Core is not classified as genotoxic according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC.


This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 474.


 



Short description of key information:
Genetic toxicity in vitro: Bacterial reversion mutation assay negative (OECD No. 471, Kr. 1)
Chromosome aberration assay negative (OECD No. 473, Kr.1)


Genetic toxicity in vitro: Gene mutation in mammalian cells negative (OECD No. 476, Kr. 1)
Genetic toxicity in vivo: Micronucleus assay negative (OECD 474, Kr. 2)        




Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The test item has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including the ATP2 draft.

Self classification:

Based on the available data, no additional classification is proposed.

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