Charcoal

Endpoint summary

Additional information

Genetic toxicity in vitro

Amestests

The three tests were performed in the tester strains TA 97a, TA 98, TA 100, TA 102, and TA 1535 using both the plate incorporation and the pre-incubation method, both with and without metabolic activation by rat S9 mix. All three tests were essentially similar in design and were conducted according to OECD guideline no. 471 and EU method B.13/14.

When tested in amounts corresponding to nominal test item concentrations of up to 5,000 µg/plate, none of the extracts did induce an increased rate of revertants in any of the tester strains, neither in the presence nor in the absence of metabolic activation. No cytotoxic effects were observed at any of the tested concentrations.

Concurrently performed negative and solvent controls as well as appropriate positive controls gave the expected results.

Accordingly, charcoal was concluded to be non-mutagenic in the tests.

 

In vitro gene mutation assay in mammalian cells

In addition, the potential mutagenicity of Probe 2 was studied in an in vitro mammalian cell gene mutation assay in mouse lymphoma L5178Y TK^+/-cells which was performed according to OECD guideline no. and EU method B.17.

Based on the results of a preliminary solubility and toxicity test, concentrations of the extract corresponding to nominal test item concentrations of 128, 320, 800, 2,000, and 5,000 µg/mL were selected for the two main experiments, assay 1 and 2.

In assay 1, a 3-h treatment, both with and without metabolic activation was utilised while in assay 2, a 3-h treatment with metabolic activation and a 24-h treatment without metabolic activation were used. The relative harmonised survival the relative total growth of the cells, cell viability, and the potential mutagenicity (5-trifluorothymidine resistance) were determined.

The extract of the test item was not cytotoxic up to and including the highest concentration of 5,000 µg/mL.

The mutation frequencies observed in both assays did not show dose-related tendencies, remained far below the relevant thresholds for positive results, and were not statistically significantly different from that of the vehicle control.

Both main assays fulfilled the validity criteria. Concurrently performed positive and negative controls gave the expected results thus demonstrating the correct functioning of the assay system.

In conclusion, the extract of Probe 2 did not induce gene mutations in cultured mouse lymphoma L5178Y TK^+/-cells under the conditions of this study, neither in the presence nor in the absence of metabolic activation.

Accordingly, the test item charcoal was concluded to be non-mutagenic under the conditions of the present study.

 

In vitro chromosomal aberrations assay

The potential genotoxicity of Probe 2 was investigated in an in vitro chromosome aberration assay in V79 cells. The assay was conducted according to OECD guideline no. 473 and EU method B.10. Based on the results of a pre-test for solubility and cytotoxicity test, concentrations of 1,250, 2,500, and 5,000 μg/mL were chosen for the main assay. The main assay consisted of two independent experiments, experiment A (3 h treatment, harvest after 20 h, both with and without metabolic activation) and experiment B (3 h treatment with and 20 h treatment without metabolic activation, harvest after 28 h).

In both experiments A and B, the test item extract did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, either in the absence or in the presence of metabolic activation, up to and including the maximum concentration.

There were no statistical differences between treatment and concurrent solvent control groups, and no dose-response relationships were noted.

No statistically significant differences between treatment and concurrent negative (vehicle) control groups and no dose-response relationships were noted.

An adequate degree of cytotoxicity was observed at the highest test concentration in both experiments.

The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in the two experiments, either in the presence or absence of metabolic activation.

The validity of the test was shown using ethyl methanesulphonate (0.4 and 1.0 μL/mL) and N-nitrosodimethylamine (1.0 μL/mL) as positive controls.

In conclusion, the test item charcoal was considered as non-genotoxic in this system.

Taken together, the results of the three Ames test, the in vitro gene mutation assay in mammalian cells, and the in vitro chromosomal aberrations assay clearly indicate that charcoal is non-mutagenic and non-genotoxic.

 

Genetic toxicity in vivo

In vivo mutagenicity testing is not needed as no positive results were observed in the battery of three in vitro genotoxicity tests (see above).

 

 

 

Short description of key information:
The potential genetic toxicity of charcoal was evaluated in a series of three bacterial reverse mutation (Ames) tests as well as in an in vitro gene mutation assay in mammalian cells and an in vitro chromosomal aberrations assay.
The tests were conducted with organic extracts of the three charcoal samples Probe 1 (C-Fix = 73.3%), Probe 2 (C-Fix=80.5%), and Probe 3 (C-Fix=88.7%). While extracts of all three test items were tested in the Ames test, the in vitro gene mutation assay in mammalian cells and an in vitro chromosomal aberrations assay were conducted with the extract of Probe 2 only.
Each extract was prepared from 250 g of the respective test item by extraction with acetone : n-hexane 50:50 (v/v). The nominal concentration of the test item in each extract was 1,000 mg/mL.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained in the three tests, the gene mutation assay in mammalian cells in vitro, and the in vitro chromosomal aberrations assay, charcoal is concluded to be non-genotoxic. Accordingly, charcoal is not classified as a germ cell mutagen according to the current EU-CLP Regulation. However, results from specific mutagenicity studies in germ cells are lacking.