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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint 3 in vitro tests, performed according to OECD test guidelines, under GLP conditions, and QA, were selected as key studies:

- a) A Bacterial Reverse Mutation Assays (Ames test), performed according to OECD 471, in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). Activated Carbon - Low Density Skeleton (Chemically Activated Carbon) did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 5 Salmonella typhimurium test strains (TA1535, TA1537, TA98, TA100, and TA102), both in the absence and presence of S9-metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate. These results were confirmed in an independently repeated experiment. It is concluded that Activated Carbon - Low Density Skeleton is not mutagenic.

- b) An in vitro Mammalian Chromosome Aberration Test, performed according to OECD 473, using cultured peripheral human lymphocytes. Activated Carbon - High Density Skeleton did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in 3 independently repeated experiments, after various exposure times, at any of the concentrations tested. No effects of Activated Carbon - High Density Skeleton on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore, Activated Carbon - High Density Skeleton is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

- c) An in vitro Mammalian Cell Gene Mutation Test, performed according to OECD Guideline 476, using the mouse lymphoma thymidine kinase locus using the cell line L5178Y. No substantial and reproducible dose dependent increase in mutant colony numbers/mutation frequency was observed in both main experiments up to the maximum concentration of 130 μg/mL, with and without metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. 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, Activated Carbon - High Density Skeleton is considered to be non-mutagenic in this mouse lymphoma assay.

No supporting studies were available.

Short description of key information:
- Gene mutation in bacteria (Bacterial Reverse Mutation Assay/Ames): not mutagenic (OECD 471, GLP).
- In vitro Mammalian Chromosome Aberration Test: non-clastogenic (OECD Guideline 473, GLP). Read Across
- In vitro Mammallian Cell Gene Mutation Test: non-mutagenic ((OECD Guideline 476, GLP). Read Across

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 14, 2010 - April 29, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
performed under GLP
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His-gene: Amino acid histidine
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: TA 1537: his C 3076; rfa-; uvrB-; TA 98: his D 3052; rfa-; uvrB-; R-factor; TA 1535: his G 46; rfa-; uvrB-; TA 102: his G 428; rfa-; uvrB+; R-factor; TA 100: his G 46; rfa-; uvrB-; R-factor.
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar rat liver S9 induced by Phenobarbital/B-Naphthoflavone
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I (plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiment II (pre-incubation test): 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Strain TA 100 and TA 1535: sodium-azide; Strain TA 1537 and TA 98: 4-nitro-o-phenylene-diamine; Strain TA 102: methyl methane sulfonate; With metabolic activation: All strains: 2-aminoanthracene.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Selection time (if incubation with a selection agent): at least 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*8

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean and Standard Deviation
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in the untreated control of strain TA 102 with S9 mix were slightly above the laboratory's historical control range. Since this deviation is rather small, and no difference with the concomittent vehicle controls was present, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Chemically Activated Carbon is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The test item, Chemically Activated Carbon, was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000μg/plate. Experiment II: 33; 100; 333; 1000; 2500; and 5000μg/plate. The plates incubated with the test item showed normal background growth up to 5000μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five test strains was observed following treatment with Chemically Activated Carbon at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Chemically Activated Carbon is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 01, 2010 - May 05, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test was conducted according to OECD Test Guideline No. 473, 1997, under GLP Standards, and QA. The study was assigned a Klimisch 2 rating due to the read-across purpose, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Justification for type of information:
The justification for read across is attached as a separate document to the "Toxicological information" summary
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium, mixture 1:1)
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar rat liver S9 induced by Phenobarbital/B-Naphthoflavone
Test concentrations with justification for top dose:
In the preliminary cytotoxicity test (with and without S9 mix): 0.8, 1.5, 2.6, 4.5, 7.9, 13.9, 24.3, 42.4, 74.3, and 130.0 ug/mL.
In the cytogenetic experiments:
- Without S9 mix:
Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL
Experiment IIA (22 hrs continuous exposure): 10.7, 18.7, and 32.7 ug/mL
Experiment IIB (22 hrs continuous exposure): 13.9, 24.3 , and 42.4 ug/mL
- With S9 mix:
Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL
Experiment IIA (4 hrs exposure): 2.0, 3.5, and 6.1 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix: ethylmethane sulfonate; with S9 mix: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I: (± S9-mix): 4 hours
Experiment IIA: + S9-mix: 4 hours; - S9-mix: 22 h continuous exposure
Experiment IIB: - S9-mix: 22 h continuous exposure.
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 μg/ml medium, last 3 hours of incubation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures for each treatment

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

OTHER: Dose Selection: with respect to the molecular weight and the purity of the test item, 130.0 ug/mL of Steam Activated Carbon (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 0.8 and 130.0 ug/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed at the end of treatment at 4.5 ug/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, 130.0 ug/mL was chosen as top treatment concentration in the absence of S9 mix and 24.3 ug/mL in the presence of S9 mix. Due to a technical error the top concentrations applied in Experiment IIA were 100 ug/mL in the absence of S9 mix and 18.7 ug/mL in the presence of S9 mix. Since the highest required concentration was not obtained in the absence of S9 mix, this experimental part was repeated with a top concentration of 130.0 ug/mL.
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data, and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
The assay can indicate an aneugenic potential of the test item if:
- the number of induced numerical aberrations is not in the range of the historical control data.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 4.5 ug/mL and above in the absence and presence of S9 mix. In Experiment IIA precipitation occurred at 32.7 ug/mL and above in the absence of S9 mix and at 6.1 ug/mL and above in the presence of S9 mix. In Experiment IIB in the absence of S9 mix precipitation occurred at 42.4 ug/mL and above.
The evaluated experimental points in the cytogenetic experiments were as follows: without S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (22 hrs continuous exposure): 10.7, 18.7, and 32.7 ug/mL; Experiment IIB (22 hrs continuous exposure): 13.9, 24.3 , and 42.4 ug/mL. With S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (4 hrs exposure): 2.0, 3.5, and 6.1 ug/mL..

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration rates of the cells after treatment with the test item (0.5 – 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps), and within the range of the laboratory´s historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions reported, the test item Steam Activated Carbon did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Steam Activated Carbon is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item Steam Activated Carbon, suspended in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix, in accordance with OECD Guideline 473. Three independent experiments were performed. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 130.0 ug/mL (approx.10 mM) was chosen, considering the molecular weight and the purity of the test item. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. The evaluated experimental points in the cytogenetic experiments were as follows: without S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (22 hrs continuous exposure): 10.7, 18.7, and 32.7 ug/mL; Experiment IIB (22 hrs continuous exposure): 13.9, 24.3 , and 42.4 ug/mL. With S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (4 hrs exposure): 2.0, 3.5, and 6.1 ug/mL.

In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item Steam Activated Carbon did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Steam Activated Carbon is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 16, 2010 - June 07, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test was conducted according to OECD Test Guideline No. 476, under GLP Standards, and QA .The study was assigned a Klimisch 2 rating due to the read-across purpose, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Justification for type of information:
The justification for read across is attached as a separate document to the "Toxicological information" summary
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
Not aplicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Male Wistar rat liver induced by Phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Both main experiments with and without metabolic activation: 2.0; 4.1; 8.1; 16.3; and 130 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 10 % deionised water (± S9 mix)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: Methyl Methane Sulfonate; +S9 mix: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours; Experiment II: -S9 mix: 24 hours; +S9 mix: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 10*6

DETERMINATION OF CYTOTOXICITY
- Method: the pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 1.0 μg/mL and 130 μg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity and the molecular weight of the test item.

OTHER EXAMINATIONS: DETERMINATION OF SIZE DISTRIBUTION OF THE COLONIES, as small TK-/- colonies may result from chromosomal damage (aberrations) to the TK locus and adjacent genes: colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10*6 cells above the corresponding solvent control. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
Statistics:
The survival rate and viability were determined based on the Poisson distribution method. The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions. A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see also additional information on results
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
see also additional information on results
Vehicle controls validity:
other: yes, see additional information on results
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both experiments precipitation was observed at 16.3 μg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Since no relevant cytotoxicity occurred, the maximum concentration of experiments I and II was again 130 μg/mL equal to approximately 10 mM. The next lower concentration was 16.3 μg/mL which was the lowest precipitating concentration as judged by the unaided eye. The lower concentrations were spaced by a factor of 2.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

SOLVENT CONTROLS: Positive controls gave the appropriate results.

DETERMINATION OF SIZE DISTRIBUTION OF THE COLONIES: No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

No substantial and reproducible dose dependent increase in mutant colony numbers/mutation frequency was observed in both main experiments up to the maximum concentration of 130 μg/mL, with and without metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. 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, Steam Activated Carbon is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

The study was performed in accordance with OECD Guideline 476 to investigate the potential of Steam Activated Carbon 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 metabolic 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. Both main experiments were evaluated at the following concentrations with and without metabolic activation: 2.0; 4.1; 8.1; 16.3; and 130 μg/mL. In both experiments precipitation was observed at 16.3 μg/mL and above with and without metabolic activation.

No relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment. No substantial and reproducible dose dependent increase in mutant colony numbers/mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

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, Steam Activated Carbon is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The justification for read across is attached
Reason / purpose for cross-reference:
read-across source
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 4.5 ug/mL and above in the absence and presence of S9 mix. In Experiment IIA precipitation occurred at 32.7 ug/mL and above in the absence of S9 mix and at 6.1 ug/mL and above in the presence of S9 mix. In Experiment IIB in the absence of S9 mix precipitation occurred at 42.4 ug/mL and above.
The evaluated experimental points in the cytogenetic experiments were as follows: without S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (22 hrs continuous exposure): 10.7, 18.7, and 32.7 ug/mL; Experiment IIB (22 hrs continuous exposure): 13.9, 24.3 , and 42.4 ug/mL. With S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (4 hrs exposure): 2.0, 3.5, and 6.1 ug/mL..

COMPARISON WITH HISTORICAL CONTROL DATA: The aberration rates of the cells after treatment with the test item (0.5 – 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 1.5 % aberrant cells, excluding gaps), and within the range of the laboratory´s historical solvent control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
A chromosomal aberrations study was performed on Steam Activated Carbon, and the results are used for Read Across to AC-LDS.

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions reported, the test item Steam Activated Carbon did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Steam Activated Carbon is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.
Executive summary:

The test item Steam Activated Carbon, suspended in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix, in accordance with OECD Guideline 473. Three independent experiments were performed. In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 130.0 ug/mL (approx.10 mM) was chosen, considering the molecular weight and the purity of the test item. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. The evaluated experimental points in the cytogenetic experiments were as follows: without S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (22 hrs continuous exposure): 10.7, 18.7, and 32.7 ug/mL; Experiment IIB (22 hrs continuous exposure): 13.9, 24.3 , and 42.4 ug/mL. With S9 mix: Experiment I (4 hrs exposure): 1.5, 2.6, and 4.5 ug/mL; Experiment IIA (4 hrs exposure): 2.0, 3.5, and 6.1 ug/mL.

In this study, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by clearly reduced mitotic indices could be observed. In both experiments, in the absence and presence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item Steam Activated Carbon did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, Steam Activated Carbon is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The justification for read across is attached
Reason / purpose for cross-reference:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
see also additional information on results
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
see also additional information on results
Vehicle controls validity:
other: yes, see additional information on results
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both experiments precipitation was observed at 16.3 μg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Since no relevant cytotoxicity occurred, the maximum concentration of experiments I and II was again 130 μg/mL equal to approximately 10 mM. The next lower concentration was 16.3 μg/mL which was the lowest precipitating concentration as judged by the unaided eye. The lower concentrations were spaced by a factor of 2.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

SOLVENT CONTROLS: Positive controls gave the appropriate results.

DETERMINATION OF SIZE DISTRIBUTION OF THE COLONIES: No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.
Conclusions:
An mouse lymphoma assay was performed on Steam Activated Carbon, and the results are used for Read across to AC-LDS.

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

No substantial and reproducible dose dependent increase in mutant colony numbers/mutation frequency was observed in both main experiments up to the maximum concentration of 130 μg/mL, with and without metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. 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, Steam Activated Carbon is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

An mouse lymphoma assay was performed on Steam Activated Carbon, and the results are used for Read across to AC-LDS. The study was performed in accordance with OECD Guideline 476 to investigate the potential of Steam Activated Carbon 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 metabolic 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. Both main experiments were evaluated at the following concentrations with and without metabolic activation: 2.0; 4.1; 8.1; 16.3; and 130 μg/mL. In both experiments precipitation was observed at 16.3 μg/mL and above with and without metabolic activation.

No relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment. No substantial and reproducible dose dependent increase in mutant colony numbers/mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

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, Steam Activated Carbon is considered to be non-mutagenic in this mouse lymphoma assay.

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

Additional information

Justification for classification or non-classification

The Ames study with Activated Carbon - Low Density Skeleton and Read Across to two in vitro studies with Activated Carbon - High Density Skeleton indicate that Activated Carbon - Low Density Skeleton does not show any genotoxic potential. Therefore, it can be concluded that the substance is not mutagenic and therefore does not need to be classified for mutagenicity according to the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS) .