Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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

Additional information

Sulphuric acid, compound with graphite (SAT Graphite) is an inorganic substance consisting of graphite and intercalated sulphuric acid and sulphate. Water solubility tests have demonstrated that sulfuric acid/sulfate are leached out to a lesser extent in case aqueous extracts are prepared with SAT Graphite.

Following the explanations in ECHA´s Guidance Guidance for identification and naming of substances under REACH and CLP, ,,for a UVCB substance, all known constituents and all constituents present at concentrations ≥ 10% should be specified by at least an English-language IUPAC name and preferably a CAS number. …Constituents that are relevant for the classification and/or PBT assessment of the substance shall always be identified by the same identifiers, independently from their concentration.“ Consequently a focus will be laid on the main constituents (graphite and sulfate) of SAT Graphite regarding the assessment of any potential genotoxic effects in in vitro systems.

The result of the water solubility test reveals that also metals are leached out. These metals stem from naturally occuring minerals in which these metals are present as metal oxides and which are generally included in natural graphite as a natural impurity. Based on the low concentrations of these metals and the non-hazardous properties of these minerals, these metals will not be considered for any further assessment regarding the potential to induce genetic toxicity in in vitro systems.

 

 

1.      Studies performed

In the course of the CSA of SAT Graphite two genotoxicity tests, namely the bacterial reverse mutation assay (Ames Test) and the in vitro mammalian cell micronucleus test, have been performed under GLP conditions and in accordance to OECD/EU guidelines. Therefore they have been allocated the highest reliability (Klimish code 1). SAT Graphite is an inorganic solid. Following OECD guidelines, in case of insoluble substances, in vitro tests for genetic toxicity are carried out with extracts produced from these substances. Polar extracts of SAT Graphite (0.9 % aqueous solution of SAT Graphite extract) include the same leached-out ions, as detected in the course of the water solubility testing (see IUCLID endpoint 4.8). Neither graphite, nor sulphuric acid and sulphate (such as sodium sulphate) are known to be genotoxic substances.

In order to screen SAT Graphite for a potential of point mutation, polar and non-polar extracts of SAT Graphite have been tested in the bacterial reverse mutation assay. As expected, the results of the performed Ames test with SAT Graphite demonstrated that neither the leached out sulphuric acid and sulphate ion (detected via concentration of leached out sulphur) induced gene mutations.

Cell culture extracts of SAT Graphite (which have for example been prepared for performance of the in vitro mammalian micronucleus assay in human lymphocytes with SAT Graphite) show similar acidic properties, as assessed in the water solubility and the acidic reserve tests performed with SAT, respectively (see IUCLID endpoints 4.8 and 4.20). As expected, the in vitro mammalian micronucleus assay performed with SAT Graphite demonstrated that the leached out sulphuric acid and sulphate ions did not induce any reactions in human lymphocytes during the in vitro mammalian micronucleus assay.

As a preliminary conclusion it can be stated that the negative testing results of the two tests gave no indication of genotoxic potential of extracts of SAT Graphite, which is not surprising as the leached out substances are known not to induce genetic toxicity (see later).

 

 

2.      In vitro Mammalian Cell Gene Mutation Test

According to the preparation and testing procedure, described in the OECD Guideline No. 476 for in vitro Mammalian Cell Gene Mutation Tests, ,,solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells.” In case of water or polar extracts (i.e. aqueous NaCl-solution) as vehicle the resulting aqueous solution will include the same low concentrations of metals and ions as already detected during the performance of the water solubility test (see IUCLID endpoint 4.8) and would additionally exhibit acidic properties, as detected during the assessment of the acid reserve (see IUCLID endpoint 4.20).

One of the drawbacks of the in vitro gene mutation study in mammalian cells known is that artificial positive results might occur which actually do not reflect intrinsic mutagenic properties. These effects can for instance be caused by low pH values (COMMITTEE ON MUTAGENICITY OF CHEMICALS IN FOOD, CONSUMER PRODUCTS AND THE ENVIRONMENT (COM) - GUIDANCE ON A STRATEGY FOR TESTING OF CHEMICALS FOR MUTAGENICITY (Document from December 2000)). This fact has also been considered in the OECD Testing Guideline No. 476 by emphasising that ,,positive results which do not reflect intrinsic mutagenicity may arise from changes in pH…”.

Identical findings have furthermore been observed by Scott, D. et al. (International Commission for Protection against Environmental Mutagens and Carcinogens, Genotoxicity under extreme culture conditions. A report from ICPEMC Task Group 9, Mutation Research/Reviews in Genetic toxicology, 257, 147 - 205).

Similar observations have been made with other inorganic acids (such as hydrochloric acid) and even with weak organic acid (such as acetic acid).

 

Conclusion: Against the background that low pH values can cause false positive results, the registrants concluded that performance of the in vitro Mammalian Cell Gene Mutation Test with SAT Graphite would not lead to any gain of additional reliable information on genotoxicity, as any possibly occuring effects on mutant frequency could be pH-value related.

 

Read-across to sodium sulfate and graphite:

Exposure of biological systems to sulphuric acid results in dissociation into hydronium and sulphate ions. During performance of the mammalian cell gene mutation assay with SAT Graphite, the hydronium ions will either lead to a decrease of the pH value of the testing medium or will be buffered. In order to exclude chemical effects of acids, a prior neutralisation (e.g. with a NaOH solution or a phosphate buffer) might be applied, resulting in an assessment of mutagenic effects of the neutralisation product sodium sulphate. During the performance of the in vitro genetic toxicity study the respective target gene would consequently only be exposed the sulphate ions.

A study in accordance with OECD 476 was performed to investigate the potential of Sodium Sulphate to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The highest concentration (1420 μg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Sodium Sulphate is considered to be non-mutagenic in this mouse lymphoma assay.

In order to assess the genotoxic potential of SAT Graphite, the underlying main components – graphite – needs to be considered, too. Graphite itself can be regarded as water-insoluble - only the intercalated sulphate/sulfuric acid – can be leached out into an aqueous media (the respective extent of this process has been assessed during the water solubility test) to a particular degree. Due to the insolubility of graphite, it can be expected that the membranes of the relevant mammalian cells cannot be crossed and therefore graphite itself cannot be regarded as systemically available.

This assumptions is backed by the negative results of the in vitro gene mutation study in mammalian cells with graphite.

The in vitro mammalian cell gene mutation test was performed to evaluate the mutagenic potential of expanded graphite powder based on quantification of forward mutations at the thymidine kinase locus of mouse lymphoma L5178Y/ TK+/-cells. The study was conducted in compliance with the OECD Guideline for the Testing of Chemicals No. 476 (In Vitro Mammalian Cell Gene Mutation Test, July 21, 1997), the EU Guideline B.17, and the Principles of Good Laboratory Practice (OECD January 21, 1998 and German Chemicals Law, § 19a, Appendix 1, July 02, 2008). Preparation of the vehicle (phosphate buffer with soy lecithin) and the test item stock suspensions and characterisation of the test item suspensions were not performed under GLP but were done in a DIN EN ISO/IEC 17025 accredited laboratory.

In the concentrations used in the main experiments, expanded graphite powder did not lead to marked changes in pH and osmolality of the incubation media. The test item, both without and with S9-mix, induced some concentration-depen­dent reduction in viability, as judged by suspension growth (SG) and relative total growth (RTG). In contrast, plating efficiency (PE) and relative survival (RS) of survivor II plates were not altered significantly in the absence of S9-mix, compared to the vehicle control. With S9-mix, there was a slight, not concentration-dependent reduction in RS in 4 out of 5 concentrations, with a maximal reduction to 74.9 % at 500 µg/ml of the test item. The negative and vehicle controls exhibited accep­table mutant frequencies (MF), which were within the normal range (50 - 200 mutants per 106viable cells; Moore et al., 2003) for L5178Y/ TK+/-cells at the TK+/-locus. The positive controls MMS (-S9-mix) and CP (+S9-mix) in­duced a marked increase in mean MF, indicating satisfactory perfor­mance of the test and sufficient activity of the metabo­lising system. Based on the concept of the "Global Evaluation Factor" (Moore et al., 2003) expanded graphite powder, in the concen­tration range tested, did not induce any relevant increase in mean MF (both- and +S9-mix). However, slight enhancement of MF was noted in each case at 500 and 1000 µg/ml. Nevertheless all test item-treated cultures, except 1000 µg/ml (+S9-mix, MF = 204.5) exhibited MFs, which were within the normal range for negative/vehicle controls (see above) and are thus considered biological irrelevant. Mutations most likely represent spontaneous mutations and/or mutations based on unspecific particle-like effects.

 

As a conclusion it can therefore be stated that both with and without S9-mix, expanded graphite powder did not induce a relevant, clearly dose-dependent increase in the mean frequency of TFT-resistant mutants (MF) in the concentration range and under the test conditions used. Thus, under the conditions and restrictions of this assay, expanded graphite powder did not show evidence of substance-specific induction of gene mutations in mouse lymphoma L5178Y/TK+/- cells (the mammalian cell model used).

 

Overall conclusion:

SAT Graphite is an inorganic substance consisting of graphite and intercalated sulphuric acid and sulfate. Water solubility tests have demonstrated that SAT is insoluble but sulphuric acid/sulfate can beleached out in case aqueous extracts are prepared with SAT Graphite.

To provide some basic information in the registration dossier two genotoxicity tests, namely the bacterial reverse mutation assay (Ames Test) and the in vitro mammalian cell micronucleus test have been conducted with extracts of SAT Graphite although considering the substances leached out from SAT Graphite it was clear that effects could not be expected. Against this background it was not astonishing that the results of these tests have been negative.

In case the registrant would perform the In vitro Mammalian Cell Gene Mutation Test, following the relevant OECD guideline, water based extracts prepared from SAT Graphite will be used for testing. Graphite itself is insoluble and would therefore not be present in the extract. Furthermore, graphite has been shown to be not mutagenic in the In vitro Mammalian Cell Gene Mutation Tests. Based on the results of the water solubility study, leached out sulphuric acid and sulphate ions can be expected in the water based extract. The outlined weight-of-evidence approach has provided a sound data basis which proves that the leached-out ions – hydronium, sulphate, – exhibit no genotoxic potential. It is therefore foreseeable that the result of an In vitro Mammalian Cell Gene Mutation Test with extracts prepared from SAT Graphite will be negative. Even in case a positive result would be achieved, this would not allow any conclusion regarding mutagenicity as this positive result could be caused by the low pH value of the extract.

The registrants are therefore convinced that there is enough weight of evidence to conclude that SAT Graphite is not mutagenic in terms of the in vitro gene mutation study in mammalian cells.

 

 

 

 

 

 

 

 

 

 

 

 

Justification for classification or non-classification