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Genetic toxicity in vitro

Description of key information

No genetic toxicity studies with naphthenic acids, zinc salts, basic are available, thus the genetic toxicity will be addressed with existing data on the moieties liberated upon dissolution, zinc and naphthenic acids.

Naphthenic acids, zinc salts, basic is not expected to be genotoxic, since the two moieties zinc and naphthenic acid (as calcium and sodium salts of naphthenic acids and salts of zinc in a weight of evidence approach) have not shown genotoxic potential in a range of test systems. Thus, naphthenic acids, zinc salts, basic is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments as genetic toxicant.

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

Genetic toxicity in vivo

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

Additional information

Introduction

A comprehensive data gap analysis was conducted for the entire substance portfolio of the Metal carboxylates REACH Consortium (MCRC), covering 10 metal carboxylates in total. This literature screening effort included:

 

  • all available proprietary studies from the Metal carboxylates REACH Consortium (MCRC)
  • detailed literature searches in online databases
  • screening of human health review articles
  • rigorous quality and reliability screening according to Klimisch criteria, where those criteria apply

 

During the literature search and data gap analysis it became obvious that the overall database on substance-specific human health hazard data for the metal carboxylates is too scarce to cover all REACH endpoints. Therefore, the remaining data gaps had to be covered by either experimental testing or weight of evidence from similar substances.

 

The endpoints skin sensitisation, genetic toxicity, repeat dose toxicity and reproductive toxicity are addressed by weight of evidence approach, using a combination of data on the free organic acid and the metal (or one of its readily soluble salts). This way forward is acceptable, since metal carboxylates dissociate in the organic anion and the metal cation upon dissolution in aqueous media (please refer to the water solubility data in section of the IUCLID and chapter of the CSR).

 

Since synergistic effects are not expected for the metal carboxylates, the human health hazard assessment consists of an individual assessment of the metal cation and the organic anion.

 

The hazard information of the individual constituents was obtained from lead registrants via license to use on the existing REACH registration dossiers, which were submitted to ECHA in 2010. All lead-registrant dossiers were checked for completeness and accepted by ECHA, i.e. a registration number was assigned.

 

Naphthenic acid, zinc salts is the zinc metal salt of naphthenic acid, which readily dissociates to the corresponding metal zinc cation and naphthenate anions. The zinc cation and the naphthenic acid anion are considered to represent the overall toxicity of the naphthenic acid, zinc salts in a manner proportionate to the free acid and the metal (represented by one of its readily soluble salts). The neutral and basic form of naphthenic acid, zinc salt are merely distinguished by their zinc content (min. 12%, max. 20%). This difference in zinc content is not believed to alter the overall toxicity of zinc naphthenate.

 

Although the term „constituent“ within the REACH context is defined as substance (also being part of a mixture), the term constituent within this hazard assessment is meant to describe either part of the metal carboxylate salt, i.e. anion or cation.

Genetic toxicity

No genetic toxicity study with naphthenic acid, zinc salts (neutral/basic) is available, thus the genetic toxicity will be addressed with existing data on the dissociation products as detailed in the table below.

 

Table: Summary of genetic toxicity data of the naphthenic acid, zinc salts (neutral/basic) and the individual constituents.

 

(slightly soluble) zinc substances

Naphthenic acid

Naphthenic acid, sodium and calcium salts

In vitro gene mutation in bacteria

negative

(weight of evidence)

negative

negative
(read-across)

In vitro cytogenicity in mammalian cells or in vitro micronucleus test

negative

negative
(read-across)

In vitro gene mutation study in mammalian cells

negative

negative
(read-across)

In vivo cytogenicity

negative

negative
(read-across)

 

Zinc

Severalin vitro studies and two in vivo studies are available on the genotoxicity of zinc oxide. Data on other zinc compounds have also to be taken into account, as the basic assumption is made that after intake all zinc compounds (including metallic zinc) are changed (at least in part) to the ionic species and that it is this zinc cation that is the determining factor for the biological activities of the zinc compounds.

The genotoxicity of soluble and slightly soluble zinc compounds have been extensively investigated in a wide range ofin vitroandin vivostudies. Thein vitroinvestigations included non-mammalian and mammalian test systems covering the endpoints of gene mutation, chromosomal aberrations, sister chromatide exchange, unscheduled DNA synthesis (UDS), as well as cell transformation. Availablein vivogenotoxicity assays included themicronucleus test andchromosomal aberration test.

The investigated zinc compounds did not increase the mutation frequencies in the majority of bacterial or mammalian cell culture systems. For example, zinc chloride, zinc sulphate, zinc bis(dihydrogen phosphate), zinc oxide or zinc monoglycerolate were consistently negative in the Ames test. While zinc chloride was also negative for gene mutations in the mouse lymphoma assays, there was some evidence that zinc oxide, zinc acetate or zinc monoglycerolate induced in the absence of metabolic activation the formation of mutation colonies. Several reviewers noted, however, that these mutations were observed at cytotoxic concentrations and that the analysis did not distinguish between big and small colonies which could be caused by gene mutation or chromosomal aberrations (Thompson et al.,1989, WHO, 2001; EU RAR, 2008; MAK, 2009).

Conflicting information was further found when zinc compounds were examined for their potential to induce chromosomal aberrations or sister chromatid exchange in mammalian cell systems or when evaluated in the cell transformation assay. Positive as well as negative results were obtained in these cell systems with either soluble or slightly soluble zinc compounds. In those studies where chromosomal aberrations or sister chromatid exchange has been observed, these were generally considered to be weak and occurred only at high, often cytotoxic concentrations. Moreover, these positive in vitro findings have also to be seen in context of the impact that changes in zinc levels can have on cell system processes that are controlled by a strict metal homeostasis. A change of this metal homeostasis due to increased zinc levels, may lead to a binding of zinc to amino acids like cysteine and therefore to an inhibition of certain enzymes. This can lead to interactions with the energy metabolism, signal transmission and apoptotic processes which can lead to the observed clastogenic or aneugenic effects in in vitro systems (EU RAR, 2008; MAK, 2009).

In addition to above mentioned in vitro investigations, various soluble and slightly soluble zinc compounds have also been studied in a range of in vivo studies including the micronucleus test, SCE and chromosomal aberration test or dominant lethal mutation assay in mice or rats as well as in the Drosophila melanogaster SLRL test. The zinc compounds were consistently negative in the micronucleus and in the assay with Drosophila melanogaster. Zinc sulphate was further negative in a dominant lethal assay in rats.

As discussed in section 5.7.1.2, equivocal and sometimes contradictory results were obtained in the in vivo chromosomal aberration assays. These equivocal finding likely a reflection of inter-study differences in routes, levels, and duration of zinc exposure, the nature of lesions scored (gaps compared to more accepted structural alterations) and great variability in the technical rigour of individual studies (WHO, 2001). The German MAK committee reviewed the existing in vivo evidence and concluded that particularly those studies indicating clastogenic effects involved a lot of methodological uncertainties which do not allow overruling those in vivo studies which did not provide any evidence for chromosomal aberrations in vivo. Moreover, the Dutch rapporteur of EU risk assessment of zinc compounds under the EU existing substance legislation considered the positive in vitro findings for chromosomal aberration and SCE assays to be overruled by the overall weight of evidence of negative in vivo tests for this endpoint (EU RAR, 2008).

The overall weight of the evidence from the existing in vitro and in vivo genotoxicity assays suggests that zinc compounds do not have biologically relevant genotoxic activity. This conclusion is in line with those achieved by other regulatory reviews of the genotoxicity of zinc compounds (WHO, 2001; SCF, 2003; EU RAR, 2008, MAK, 2009). Hence, no classification and labelling for mutagenicity is required.

 

Naphthenic acid

No experimental in vitro genetic toxicity studies were available for Naphthenic acids, however Weight of Evidence was available from ‘Sodium naphthenates’:

- Salmonella bacterial mutagenicity: negative up to >333 μg/L with and without metabolic activation (NTP, 1993; HPVIS, 2012).

- Chromosomal aberration: negative in CHO cells at 54, 116 & 250 μg/mL without metabolic activation and 25, 54, 116 & 250 μg/mL with metabolic activation (NTP, 1993; HPVIS, 2012).

- Sister Chromatid Exchange: weakly positive to positive when tested at concentrations of 17, 59, 167, 500 ug/mL (Trial 1; without metabolic activation) and 100, 150, 200, 250 µg/mL (Trial 2; without metabolic activation) and negative at 17, 59, 167, 500 µg/mL with metabolic activation (NTP, 1993; HPVIS, 2012). Although a positive result is obtained in 2 separate runs without metabolic activation, the validity of these results is questionable since the occurrence of cytotoxicity is not well documented.

 

Further Weight of Evidence is available from ‘Calcium naphthenates’:

- E-coli and Salmonella bacterial reverse mutagenicity: negative in WP2 uvr A and Salmonella TA 1535, TA 1537, TA 98 and TA 100 strains when tested at 31.25 - 4000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Saccharomyces cerevisiae: non-mutagenic when tested at 10 -5000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Rat Liver chromosomal damage: non-mutagenic at 62.5-250 μg/mL without metabolic activation (Shell Research Ltd, 1983).

- In vitro testing in L5178Y T K +/-mouse lymphoma cells both with and without metabolic activation at 0.0005 to 10000 µg/mL showed a positive effect in the absence of metabolic activation (Seifried et al, 2006), however when studying the raw data and the evaluation criteria the applicant cannot support this conclusion.

Further, QSAR calculations also show that naphthenic acids, including various molecules from C6 -C30 chain lengths, are predicted to be consistently non-mutagen using the VEGA QSAR model and Toxtree (Benigni-Bossa) rulebase for mutagenicity.

   

Finally, an in vivo micronucleus test was conducted in male and female Wistar rats with refined Naphthenic acids doses at 100, 300 and 900 mg/kg bw by oral gavage (HPVIS, 2010). A total of 1000 erythrocytes/slide were evaluated (both polychromatic PCE and normochromatic erythrocytes NCE), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal. The frequencies of micronuclei in in bone marrow did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.

 

All genotoxicity and mutagenicity tests and modelling tools revealed the absence of genotoxic and mutagenic properties for naphthenic acids. Therefore a non-classification for this endpoint is justified.

 

Naphthenic acids, zinc salts, basic

Naphthenic acids, zinc salts, basic is not expected to be genotoxic, since the two moieties zinc and naphthenic acid (as calcium and sodium salts of naphthenic acids and salts of zinc in a weight of evidence approach) have not shown genotoxic potential in a range of test systems. Thus, naphthenic acids, zinc salts, basic is not to be classified according to regulation (EC) 1272/2008 and its subsequent amendments as genetic toxicant. Further testing is not required.

Justification for classification or non-classification

Naphthenic acids, zinc salts, basic is not to be classified according to regulation (EC) 1272/2008 as genetic toxicant, since none of the constituents have shown genotoxic potential in a range of test systems.