Naphthenic acids, bismuth salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Whereas bismuth hydroxy nitrate oxide has been assessed in an in vitro mammalian gene mutation test (OECD 476), using mouse lymphoma L5178Y cells (with and without metabolic activation), and found not to induce mutation at the hprt locus of L5178Y mouse lymphoma cells, the substance bismuth salicylate was assessed for chromosomal aberrations in an in vitro assay (OECD 473) using Chinese hamster lung fibroblasts (V79), with and without metabolic activation, and was also found negative, not being clastogenic.

The substances bismuth nitrate and bismuth salicylate were assessed in Ames studies, whereas the bismuth nitrate was only tested with E.coli cells and confirmed negative, and the bismuth salicylate was assessed in salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, all with and without metabolic activation, also found being negative for mutagenicity/genotoxicity.

Thus, in summary it can be concluded that bismuth compounds are negative in four in vitro mutagenicity tests.

The second source substance naphthenic acids and its calcium salts has been investigated in a rat liver chromosomal damage test system found being non-mutagenic, in an Ames test, using E.coli WP2 and WP2 uvr A strains, as well as salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537, all with and without metabolic activation. All results were negative for mutagenicity. Furthermore, the substance was tested and found non-mutagenic in the Yeast gene conversion test system Saccharomyces cerevisiae when tested at 10 - 5000 µg/plate with and without metabolic activation.

Besides in total seven in vitro studies, all being negative for genotoxicity, it is reasonably concluded that naphthenic acids, bismuth salts, the target substance, is non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Besides the seven negative in vitro studies, naphthenic acids was tested in an in vivo chromosomal aberration test (OECD 474) with male and female Wistar rats, dosed at 100, 300 and 900 mg/kg bw by oral gavage and found negative for inducing chromosomal aberrations. This further support the negative outcome for assessing the mutagenicity of naphthenic aids, bismuth salts.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

combined weight of evidence from in vitro and in vivo studies

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substance naphthenic acids, bismuth salts is manufactured from bismuth oxide/hydroxide and 3 equivalents of naphthenic acids, resulting in the bismuth tri-naphthenate. Thus, to assess mutagenicity/genotoxicity, results for bismuth 3+ cations were assessed as well as data for naphthenic acids, the two potential hydrolysis products of the substance. The undissociated substance is considered uncritical, as its molecular mass of ~850 Dalton makes it unlikely that such compounds efficiently pass biological membranes. Thus, the mutagenicity/genotoxicity on naphthenic acids, bismuth salts will be dominated by its ions, derived from hydrolysis.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source compound naphthenic acids is the starting material for the manufacturing of naphthenic acids, bismuth salts. Naphthenic acids do contain mainly hydrocarbon acids with a carbon range from 10 - 15 (other naphthenic acids may have wider ranges), with a variable number of cyclics contained (n = 0, 1, 2 and rarely 3). To a minor extent also aliphatics may be present as minor "impurities".
The naphthenic acids are reacted in a slight excess of >3 equivalents of naphthenic acids with bismuth oxide to derive naphthenic acids, bismuth salts, the target substance, containing bismuth tri-naphthenate and a slight excess of naphthenic acids. Thus, the starting compound for the synthesis of the target compound is actually the source substance. Whereas the bismuth oxide used has a purity of 99% by weight typically, the naphthenic acids, being a UVCB-type substance, are of 100% purity, by definition.
The source substances used to assess the bismuth genotoxicity properties are soluble bismuth compounds, namely bismuth hydroxide nitrate oxide, bismuth nitrate and bismuth salicylate. Being soluble forms of bismuth(III) compounds, these substances are suitable to achieve high Bi3+ concentrations in aqueous biotic systems; therefore, these substances were chosen for assessing mutagenicity/genotoxicity properties as surrogate for bismuth compounds, as it allowed achieving high Bi3+ concentrations and thus a maximum bioavailability, compared to bismuth oxide, being hardly soluble and hardly absorbed.
Thus, the two source compounds naphthenic acids and naphthenic acids, calcium salts (as surrogate for naphthenate anions) and bismuth hydroxy nitrate oxide, bismuth nitrate and bismuth salicylate (as surrogate for bismuth cations) are appropriate surrogates for assessing the mutagenicity/genotoxicity of the target substance.

3. ANALOGUE APPROACH JUSTIFICATION
Although the two source substances, naphthenic acids and corresponding calcium salts as well as bismuth hydroxide nitrate oxide/bismuth nitrate/bismuth salicylate, are different in respect of properties compared to the target substance, the read-across is justified, as the source substance bismuth hydroxy nitrate oxide/bismuth nitrate/bismuth salicylate allows to achieve a higher bismuth concentration in aqueous systems and thus better absorption, compared to the target substance (water solubility of < 0.036 mg/L).
Whereas bismuth hydroxy nitrate oxide has been assessed in an in vitro mammalian gene mutation test (OECD 476), using mouse lymphoma L5178Y cells (with and without metabolic activation), and found not to induce mutation at the hprt locus of L5178Y mouse lymphoma cells, the substance bismuth salicylate was assessed for chromosomal aberrations in an in vitro assay (OECD 473) using Chinese hamster lung fibroblasts (V79), with and without metabolic activation, and was also found negative, not being clastogenic.
The substances bismuth nitrate and bismuth salicylate were assessed in Ames studies, whereas the bismuth nitrate was only tested with E.coli cells and confirmed negative, and the bismuth salicylate was assessed in salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, all with and without metabolic activation, also found being negative for mutagenicity/genotoxicity.
Thus, in summary it can be concluded that bismuth compounds are negative in four in vitro mutagenicity tests.
The second source substance naphthenic acids and its calcium salts has been investigated in a rat liver chromosomal damage test system found being non-mutagenic, in an Ames test, using E.coli WP2 and WP2 uvr A strains, as well as salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537, all with and without metabolic activation. All results were negative for mutagenicity. Furthermore, the substance was tested and found non-mutagenic in the Yeast gene conversion test system Saccharomyces cerevisiae when tested at 10 - 5000 µg/plate with and without metabolic activation.
Finally, naphthenic acids was tested in an in vivo chromosomal aberration test (OECD 474) with male and female Wistar rats dosed at 100, 300 and 900 mg/kg bw by oral gavage and found negative for inducing chromosomal aberrations.
Thus, it can be concluded, that the bismuth(III) cations are negative in three in vitro genotoxicity studies and also the naphthenic acids and their salts, respectively were negative in three in vitro and one in vivo study and as a result, naphthenic acids, bismuth salts are also considered negative for genotoxicity.

4. DATA MATRIX
The source substances naphthenic acids and corresponding calcium salts as well as bismuth hydroxide nitrate oxide/bismuth nitrate/bismuth salicylate were chosen to assess the genotoxic potential of naphthenic acids, bismuth salts, the target substance. The idea was to choose surrogates with a maximum solubility to simulate maximum exposure and enhanced cell permeation, compared to assessing the insoluble bismuth tri-naphthenate (main component of the target substance). Furthermore, the target substance does contain residual free naphthenic acids from the production process and thus naphthenic acid data are contributing as such to the genotoxic assessment of the substance.
Besides in total seven in vitro studies, all being negative for genotoxicity, this finding was also confirmed by an in vivo study with naphthenic acids. Thus, it is reasonably concluded that naphthenic acids, bismuth salts, the target substance, is non-mutagenic.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the above summarises the findings from in vitro and in vivo genetic toxicity studies

Additional information on results:

The results shown above do summarise the weight of evidence approach which is based on in total seven in vitro genetic toxicity studies and 1 in vivo genetic toxicity study, performed with surrogates for bismuth cations and naphthenic acids anions, namely naphthenic acids and its corresponding calcium salts as well as bismuth hydroxide nitrate oxide, bismuth nitrate, and bismuth subsalicylate. In none of these studies and positive or ambiguous results were found and all studies were clearly negative. Thus, in a weight of evidence approach, naphthenic acids, bismuth salts are considered non-mutagenic.

Conclusions:

The weight of evidence approach is based on in total seven in vitro genetic toxicity studies and 1 in vivo genetic toxicity study, performed with surrogates for bismuth cations and naphthenic acids anions, namely naphthenic acids and its corresponding calcium salts as well as bismuth hydroxide nitrate oxide, bismuth nitrate, and bismuth subsalicylate and none of these studies showed any positive or ambiguous results, but all were found were clearly negative. Thus, in a weight of evidence approach, naphthenic acids, bismuth salts is considered non-mutagenic.

Executive summary:

The source substances naphthenic acids and its corresponding calcium salts as well as bismuth hydroxide nitrate oxide, bismuth nitrate, and bismuth salicylate were chosen to assess the genotoxic potential of naphthenic acids, bismuth salts, the target substance. The idea was to choose surrogates with a maximum solubility to simulate maximum exposure and enhanced cell permeation, compared to assessing the insoluble bismuth tri-naphthenate (main component of the target substance). Furthermore, the target substance does contain residual free naphthenic acids from the production process and thus naphthenic acid data are contributing as such to the genotoxic assessment of the substance.

Besides in total seven in vitro studies, all being negative for genotoxicity, this finding was also confirmed by an in vivo study with naphthenic acids. Thus, it is reasonably concluded that naphthenic acids, bismuth salts, the target substance, is non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on seven in vitro and one in vivo mutagenicity studies, all being negative with and without metabolic activation, in a weight of evidence approach it is concluded that naphthenic acids, bismuth salts are non-mutagenic and thus, not classifiable for mutagenicity/genotoxicity according to CLP (Regulation EC No. 1272/2008).