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Genetic toxicity in vitro

Bacterial reverse mutation test:

Verspeek-Rip (2012) performed an Ames study (OECD 471 and EU B.13/14) with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A, in the presence and absence of metabolic activation. The test included two separate experiments.

Following test concentrations were applied in triplicate:

- Experiment 1: in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98 at 100, 333, 1000, 3330 and 5000 μg/plate

- Experiment 2: in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA at 100, 333, 1000, 3330 and 5000 μg/plate.

Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence of S9-mix (first and second experiment) and in the presence of S9-mix (second experiment). The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that barium nitrate is not mutagenic in the Ames test.

Chromosome Aberration test:

In the framework of the US National Toxicology program, an in vitro mammalian Chromosome Aberration test was performed in Chinese hamster Ovary (CHO) cells with the read-across substance barium chloride dihydrate. The test was performed according to OECD guideline 473.

Two experiments were performed using different test concentrations with and without S9 activation:

- Trial 1, without S9: 50, 160, 500 and 1600 µg/mL
- Trial 1, with S9: 50, 160, 500, 1600 and 5000 µg/mL
- Trial 2, without S9: 100, 250, 500, 1000, 1500 and 2000 µg/mL
- Trial 2, with S9: 500, 1600, 3000, 4000 and 5000 µg/mL

Cells cultured without S9 were exposed to the test item for 10 hours. Cells cultured with S9 exposed to the test item for 2 hours.

Positive and vehicle controls were used and considered valid.

It was concluded that the in vitro chromosome aberration test in CHO cells of barium chloride dihydrate was negative with and without metabolic activation.

This study was considered reliable with restrictions (K2) to reflect the read-across status.The read across justification is added in Section 13 of IUCLID.

In vitro Mouse lymphoma test:

Lloyd (2010) evaluated the mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique. Cells were exposed to barium chloride dihydrate in two separate experiments.The vehicle was purified water. Experiment I was performed using a 3 -hour treatment incubation and Experiment II was performed using 3- and 24 -hour treatment incubations. In the cytotoxicity range-finding experiment, concentrations were tested ranging from 65.06 to 2080 µg/mL (3-hour treatment; -/+ S9 and from 8.133 to 2082 µg/mL (24 -hour treatment; - S9 mix) mix).

Concentrations selected for the Experiments I and II were based on the results of this cytotoxicity range-finding experiment. Two experiments were performed with selected concentrations:

- In Experiment I concentrations ranging from 250 to 2082 µg/mL were tested in the absence and presence of S9 mix.

- In Experiment II (3 -hour treatment) concentrations ranging from 100 to 1400 µg/mL in the absence of S9 mix and from 200 to 1400 µg/mL in the presence of S9 were tested. Additionally cells were tested in a 24 -hour treatment with concentrations ranging from 100 to 1400 µg/mL in the absence of S9 mix.

Negative (vehicle) and positive controls treatments were included in each mutation experiment. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutations were induced by the positive control chemicals methyl methane sulphonate (without S9) and Benzo[a]pyrene (with S9). Therefore the study was accepted as valid.

In Experiment I and II, the mutant frequency of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor, indicating a negative result. Statistically significant linear trends were observed in Experiment II in the presence of S9 mix (3-hour treatment) and in the absence of S9 mix (24 -hour treatment). However, in the absence of any marked increases in mutant frequency under either treatment condition, these observations were not considered biologically relevant.

This study was considered reliable with restrictions (K2) to reflect the read-across status. The read across justification is added in Section 13 of IUCLID.

Other in vitro studies with barium nitrate:

- Kubo (2002) performed a non-reliable non-GLP Ames study (K3) according to a method similar to OECD Guideline 471 but only 2 strains were tested. Under the experimetnal conditions of this test, barium nitrate was not mutagenic in the presence and absence of metabolic activation.

- Oliver (1987) performed a non standard (K3) SOS chromotest. No negative nor positive control substances were tested. Limited information on methods and results were reported. Barium nitrate was observed to be negative for genotoxicity in the SOS chromotest with and without metabolic activation.

Other supportive studies:

Other studies with the read-across susbtance are available. These studies are not used in the genotoxicity assessment of the barium nitrate as other more reliable studies exists.

- Monaco (1990) performed a test similar to OECD Guideline 471 "Bacterial Reverse Mutation Test" with barium chloride. However it was not well documented and not all the strains were tested with vehicle controls in presence of S9. Barium chloride did not show mutagenic activity.

- Nishioka (1975) published the results of the mutagenic potential of barium chloride in bacteria. This K3 (non-GLP) study used B. subtilis in a recombination assay. Limited information was reported. Barium chloride tested negative in a recombination assay in wild and recombination-deficient strains of Bacillus subtilis.

- In the framework of the US National Toxicology program (1994), a mammalian cell gene mutation assay was performed according to a method similar to the OECD guideline 476. The results of the study were ambiguous.

Genetic toxicity in vivo

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required when no positive results are obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.

Three in vitro studies are available: one K1 Ames test (Verspeek-Rip, 2010) with barium nitrate and two studies with the read-across substance barium dichloride dihydrate (in vitro Chromosome Aberration and in vitro Mouse lymphoma). These two studies were considered adequate for read-across (justification is added in Section 13 of IUCLID). Overall it can be concluded that barium nitrate does not induce gene mutations in vitro in bacteria as somatic mammalian cells. Therefore the conduct of in vivo gene mutation experiments are not required.

An in vivo supportive study (Yesilada, 2001) was performed in Drosophila flies. The results were inconclusive. The study is not considered in this assessment as it was not performed in mammalian


Justification for selection of genetic toxicity endpoint
One study can not be selected as there are 3 in vitro key studies available: one with barium nitrate and 2 with the read-across substance barium dichloride dihydrate.

Short description of key information:
Genetic toxicity in vitro:
A K1 bacterial reverse mutation assay (Verspeek-Rip, 2012) was performed according to OECD guideline 471 and EU Method B.13/14 with barium nitrate in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A. The test item was not mutagenic in the presence and absence of metabolic activation.
A K2 in vitro chromosome aberration test was performed with a method similar to the OECD 473 Guideline (National Toxicology Program, 1994) with the read-across substance barium chloride dihydrate. The in vitro chromosome aberration test in CHO cells with barium chloride dihydrate was negative with and without metabolic activation.
A K2 in vitro mouse lymphoma test was performed according to the OECD 476 Guideline (Lloyd, 2010) with the read-across substance barium chloride dihydrate. It is concluded that Barium chloride dihydrate did not induce mutation at the TK locus of L5178Y mouse lymphoma cells in the absence and presence of a rat liver metabolic activation system (S9 mix).

Genetic toxicity in vivo: no study required

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data and according to the criteria of the DSD and CLP regulation, barium nitrate should not be classified for mutageniticity.