reaction mass of neodymium carbonate and praseodymium carbonate

Endpoint summary

Additional information

Regarding the mutagenicity tested in bacteria (i.e. Ames test), there was no study available on the reaction mass of neodymium carbonate and praseodymium carbonate. However, several studies were available on both constituents of the reaction mass as well as on 3 analogous substances (i.e. dicerium tricarbonate, neodymium oxide, praseodymium(III,IV) oxide). As the reaction mass, its constituents and analogues showed similar physico-chemical properties, data on constituents and analogues were thus used in a weight-of-evidence approach.

 

First, an in vitro gene mutation study in bacteria was available on dineodymium tricarbonate (Thompson P.W., 2010), the main constituent of the reaction mass (~79%). In this study (OECD 471, GLP), scored as Klimisch 2 and flagged as weight of evidence, the mutagenic potential of the test item was assessed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA. The different bacterial strains were treated with suspensions of dineodymium tricarbonate at 50, 150, 500, 1500 and 5000 µg/plate, using both the Ames plate incorporation and pre-incubation methods, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 mix).  

The sensitivity of the assay and the efficacy of the S9 mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A fine particulate precipitate was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Therefore, dineodymium tricarbonate was considered to be non-mutagenic under the conditions of this test.

 

A second Ames test was available on dipraseodymium tricarbonate (Thompson P.W., 2013), the minor constituent of the reaction mass (~21%). This study (OECD 471, GLP), scored as Klimisch 2 and flagged as weight of evidence, was performed as described above for dineodymium tricarbonate. The dose levels assessed were 50, 150, 500, 1500 and 5000 µg/plate.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test material film (opaque in appearance) was noted at and above 1500 μg/plate with an associated precipitate observed at 5000 μg/plate. Neither of these observations prevented the scoring of revertant colonies. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.  

Therefore, dipraseodymium tricarbonate was also considered to be non-mutagenic under the conditions of this test. 

 

These results were further corroborated by three other studies (OECD 471 / EU B.13/14, GLP) on analogues of the reaction mass (i.e. dicerium tricarbonate, neodymium oxide, praseodymium(III,IV) oxide). These three studies were awarded a reliability score of 2 (Klimisch, 1997), since used as read-across, and were flagged as supporting study (Wollny H.E., 2006b; Haddouk H., 2007a and 2007b). Dicerium tricarbonate was tested in S. typhimirium at 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with or without metabolic activation. The mutagenic potential of neodymium oxide (S. typhimirium, E.coli) and praseodymium(III,IV) oxide (S. typhimirium) was assessed at similar concentrations: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate in the absence or presence of metabolic activation. All studies showed negative results in the gene mutation assay in bacteria performed in absence or presence of metabolic activation.

 

The genotoxic potential of the reaction mass of neodymium carbonate and praseodymium carbonate was further investigated by performing a cytogenicity study in mammalian cells (i.e. chromosome aberration test) directly with the reaction mass. Conducted in compliance with GLP and according to the OECD guideline 473, this study was awarded a reliability score of 1 (Klimisch, 1997) and was flagged as key study (Sire G., 2013). The test item, suspended in water for injections, was tested at concentrations from 39.06 to 5000 µg/mL (act. ingr.) in two independent experiments, both with and without a liver metabolizing system. Lymphocyte cultures were exposed to the test or control items for 3 hours (with S9 mix) and 3, 20, or 44 hours (without S9 mix) then rinsed. Following exposure to the test or control items with S9 mix, the cultures were incubated in fresh medium at +37°C until harvest 20 and 44 hours after the beginning of treatment.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria; the study was therefore considered to be valid. A precipitate was observed in the culture medium at the end of the treatment periods, at dose levels ≥ 625 µg/mL. Moreover, a slight to moderate cytotoxicity was observed at dose-levels ≥ 312.5 µg/mL with and without S9 mix. However, no relevant increase in the frequency of cells with structural chromosomal aberrations was noted up to 1250 µg/mL after the 3-, 20- and 44-hour treatments with or without S9 mix.

Consequently, under the experimental conditions of this study, the reaction mass of neodymium carbonate and praseodymium carbonate was considered to cause no cytogenicity.

 

These data were further corroborated by two other cytogenicity studies (OECD 473 / EU B.10, GLP) performed on analogues of the reaction mass (i.e. dicerium tricarbonate and praseodymium(III,IV) oxide). These two studies were awarded a reliability score of 2 (Klimisch, 1997), since used as read-across, and were flagged as supporting study (Schultz M., 2006; Bowles A., 2013). Dicerium tricarbonate was tested at concentrations ranging from 45.6 to 7020 µg/mL with or without metabolic activation. The cytogenicity potential of praseodymium(III,IV) was assessed at 5, 10, 20, 40, 80 and 160 µg/mL in the absence of presence of metabolic activation. Both chromosome aberration tests showed negative results in human lymphocytes with and without metabolic activation.

 

Moreover, a mammalian cell gene mutation test (HPRT assay) was carried out on the reaction mass of neodymium carbonate and praseodymium carbonate. Conducted in compliance with GLP and according to the OECD guideline 476, the study was awarded a reliability score of 1 (Klimisch, 1997) and was flagged as key study (Hargitai J., 2013). The potential of the reaction mass to cause gene mutation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus was tested in CHO K1 Chinese hamster ovary cells. The assay was performed in two independent experiments, each using duplicate cultures. CHO cells were exposed to the test item suspended in 1% (w/v) methyl cellulose solution at concentrations ranging from 156.25 to 5000 μg/mL for 5 or 24 hours with or without metabolic activation (i.e. S9 mix). Measurements of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7-day expression period following the treatment), and mutagenicity (colony forming ability at the end of the 7-day expression period following the treatment, in the presence of 6-Thioguanine as a selective agent) were then performed.

Based on mutation frequencies of the negative and positive controls, the overall study was considered to be valid. Regardless of the exposure durations, precipitation of the test item was detected at the end of treatments in the 156.25-5000 μg/mL concentration range with and without S9 mix in both experiments. Cytotoxicity of the test item was also observed from the concentration of 625 µg/mL in these experiments. Nevertheless, in all experiments, no biologically relevant increases in the mutation frequency of CHO K1 cells were observed at any examined concentrations with or without metabolic activation. 

In conclusion, the reaction mass of neodymium carbonate and praseodymium carbonate was considered to be non-mutagenic under the conditions of this test.

 

These results were sustained by two other studies (OECD 476 / EU B.17, GLP) on analogues of the reaction mass (i.e. dicerium tricarbonate and neodymium oxide). These two studies were awarded a reliability score of 2 (Klimisch, 1997), since used as read-across, and were flagged as supporting study. Dicerium tricarbonate was tested in chinese hamster lung fibroblasts (V79) at concentrations of 143.8, 287.5, 575, 1150 1725 and 2300 µg/mL with or without S9 mix. The mutagenic potential of neodymium oxide was assessed in CHO K1 cells at concentrations ranging from 1.64 to 3365 µg/mL in absence or presence of S9 mix. Both studies showed negative results in the HPRT assay performed in absence or presence of metabolic activation (Wollny H.E., 2006a; Morris A., 2013a). A third HPRT test was performed with praseodymium(III,IV) oxide in CHO K1 cells exposed to 39.06 to 5000 µg/mL of test item (Morris A., 2013b). The test displayed positive results in absence of metabolic activation and ambiguous results in presence of S9 mix. However, as no effects were observed in the same study performed with the reaction mass itself or in other genetic studies performed on other insoluble or soluble rare earths, this result was thus considered incidental / false positive rather than a specific effect of the substance; these ambiguous data may be due to interferences between praseodymium(III,IV) oxide and test system. Thus, this study was awarded a reliability score of 3 (Klimisch, 1997) and was flagged as disregarded study.

 

At last, an in vivo genetic study was available on neodymium oxide, an analogue of the reaction mass of neodymium carbonate and praseodymium carbonate. Performed in compliance with GLP and according to the OECD guideline 474 and used as read-across, this mammalian erythrocyte micronucleus test was awarded a reliability score of 2 (Klimisch, 1997) and flagged as supporting study (Sire G., 2011). Three groups of male and female Swiss Ico: OF1 mice (n = 5/sex and group) were administered the test material via  the intraperitoneal route at dose-levels of 187.5, 375 and 750 mg/kg/day and 500, 100 and 2000 mg/kg/day for males and females respectively, over a 2-day period. Three additional male and female mice were added in the high dose groups in order to determine the plasma level of the test material. An additional group received the vehicle under the same experimental conditions and acted as control and another group received a positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

As the mean values of MPE and the PE/NE ratio for the vehicle and positive controls were consistent with historical data, the study was considered valid. No clinical signs and no mortality were observed in males given 187.5 mg/kg/day and in females given 500, 1000 and 2000 mg/kg/day. At 375 and 750 mg/kg/day (male groups), no mortality occurred, but piloerection was observed in some animals following the second treatment. The mean MPE values in the test material treated groups were similar to those of the vehicle group indicating no effect of the test material. However, a decrease in the PE/NE ratios was observed at all tested dose-levels compared to the vehicle control. Taking into account the clinical signs observed in some animals from the high-dose group, the test material concentrations found in the plasma samples and finally the decrease in the PE/NE ratios, the systemic exposure of the animals, as well as the exposure of the bone marrow cells to the test material, were clearly demonstrated.

Nevertheless as no increase in MPE values was observed in all exposed animals, the test material was considered to cause no cytogenetic damage under the conditions of this experiment.

 

As the reaction mass of neodymium carbonate and praseodymium carbonate showed similar physico-chemical properties compared to the two constituents and three analogues, the weight-of-evidence approach demonstrated that the reaction mass itself could be considered as having no genetic toxicity potential, therefore warranting no classification for genetic toxicity according to the criteria of Annex VI of Directive 67/548/EEC, Regulation (EC) No. 1272/2008 and UN GHS.

 

Genetic toxicity studies	Reaction mass of neodymium carbonate and praseodymium carbonate	Dineodymium tricarbonate	Dipraseodymium tricarbonate	Dicerium tricarbonate	Neodymium oxide	Praseodymium(III,IV) oxide
In vitro
Bacterial Reverse Mutation Assay (Ames; OECD 471, GLP)	no data	negative ± metabolic activation	negative ± metabolic activation	negative ± metabolic activation	negative ± metabolic activation	negative ± metabolic activation
Mammalian Chromosome Aberration Test (OECD 473, GLP)	negative ± metabolic activation	no data	no data	negative ± metabolic activation	no data	negative ± metabolic activation
Mammalian Cell Gene Mutation Test (HPRT; OECD 476, GLP)	negative ± metabolic activation	no data	no data	negative ± metabolic activation	no data	ambiguous with metabolic activation positive without metabolic activation
In vivo
Mammalian Erythrocyte Micronucleus Test (OECD 474, GLP)	no data	no data	no data	no data	negative	no data

Justification for selection of genetic toxicity endpoint
Several studies done on the reaction mass and/or on its constituents or analogous substances were selected and considered together using a weight of evidence approach.
Two studies were conducted on the reaction mass of neodymium carbonate and praseodymium carbonate, in compliance with GLP and according to the OECD guidelines 473 and 476. Well described and meeting the generally acceptable scientific principles, these studies were awarded a reliability score of 1 (Klimisch, 1997) and were flagged as key study.
Moreover, two other studies, performed on both constituents of the reaction mass (dineodymium tricarbonate and dipraseodymium tricarbonate), were available. Conducted in compliance with GLP and according to the OECD guideline 471, these studies were considered in a weight-of-evidence approach to conclude on the genetic toxicity potential of the reaction mass of neodymium carbonate and praseodymium carbonate. Well described, the reports were used as read-across; therefore, the studies were flagged as weight of evidence and the maximal reliability score was decreased from 1 to 2 (Klimisch, 1997), according to Practical Guide n°6.
In addition, 7 other in vitro studies on analogues (dicerium tricarbonate, neodymium oxide and praseodymium(III,IV) oxide) showing no genetic toxicity potential were also considered in this weight-of-evidence approach. All performed in vitro according to OECD/EU guidelines and in compliance with GLP, these studies were also awarded a score 2 (Klimisch, 1997), since used as read-across, and flagged as supporting study.
At last, an in vivo study was available on analogue of the reaction mass (neodymium oxide). Conducted according to OECD guideline 474 and in compliance with GLP, this study was considered in the weight-of-evidence approach and thus awarded a reliability score of 2 (Klimisch, 1997), according to Practical Guide n°6, and flagged as supporting study.

Short description of key information:
No indication of genetic toxicity based on in vitro test results: cytogenicity in mamalian cells (i.e. chromosome aberration test) and gene mutation assay in mammalian cells (i.e. HPRT assay) performed with the reaction mass of neodymium carbonate and praseodymium carbonate as well as in bacterial reverse mutation test (Ames test) performed with both constituents of the reaction mass and analogous substances (dicerium tricarbonate, neodymium oxide and praseodymium(III,IV) oxide).
In addition, further studies of cytogenicity in mammalian cells (dicerium tricarbonate, praseodymium(III,IV) oxide) and gene mutation assays in mammalians cells (dicerium tricarbonate and neodymium oxide) as well as an in vivo micronucleus test (neodymium oxide) were also considered in a weight-of-evidence approach.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the classification criteria of Annex VI of Directive 67/548/EEC, Regulation (EC) No. 1272/2008 and UN GHS, and considering the results of the in vitro genetic toxicity studies performed on the reaction mass, and all in vitro and in vivo studies performed on its constituents or analogous substances, no classification for genetic toxicity is required for the reaction mass of neodymium carbonate and praseodymium carbonate.