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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The genotoxicity profile of benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt (registered substance/target substance) was not determined by actual genotoxicity studies. Instead, read across substances were used to predict the genotoxicity of the registered substance.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been conducted to OECD guidelines and to GLP, however, has not been fully reported. In addition, as this study is used in the context of a read across, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
arochlor induced rat liver S9 extract
Test concentrations with justification for top dose:
10, 20,40,80,120,160 ug/ml
Vehicle / solvent:
tetrahydrofuran for test material
acetone for positive control
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 16 and 40 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 metaphase cells (100 per culture) each containing 19-23 chromosomes


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

Evaluation criteria:
cells with aberrant chromosomes.
Statistics:
Fisher exact test.
Species / strain:
Chinese hamster Ovary (CHO)
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 specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate and/or cloudines were present with and without metabolic activation at concentrations of 39 µg/ml and greater.


RANGE-FINDING/SCREENING STUDIES: There was an 81% reduction in cell survival at 160 µg/ml, without metabolic activation, compared with control.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

In the initial 16h harvest there were statistically significant increases with dose in the % of aberrant cells for both activated and non activated tests. These trends were not reproducible in the repeat 16h harvest and were therefore not considered biologically significant.

Conclusions:
Interpretation of results (migrated information):
negative

The test material was not genotoxic under the conditions of this study.
Executive summary:

In a mammalian cell cytogenetics assay [chromosome aberration], CHO cell cultures were exposed to a petroleum derived calcium salt derivative at concentrations of 0, 10, 20, 40, 80, 120 and 160 µg/ml with and without S9 metabolic activation.

 

Positive controls induced the appropriate response.  There was no evidence of chromosome aberrations induced over background with the test substance.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

In a supporting study, a reverse gene mutation assay in bacteria (OECD 471), the strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to a calcium sulfonate read across substance, (analogue of CAS 70024-69-0), at concentrations of 0.1, 0.33, 1.0, 3.33 and 10 mg/plate in the presence and absence of mammalian metabolic activation (Myers et al., 1989). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate were used. No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10 mg/plate) and concentrations of S9 mix of 25-400 μL. In the absence of any effect 25 μL S9 mix/plate was used in the mutagenicity study. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were confirmed in a second independent experiment. 100 μL of test material, positive control or vehicle control were added to each plate along with 100 μL of tester strain, S9 mix (if needed) and 2.0 mL of top agar. This was overlaid onto the surface of 25 mL minimal bottom agar in a petri dish. Plates were incubated for 48 hours at 37 °C. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However ,after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However, since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the pre-incubation modification of the Salmonella/microsome test.

In a supporting study, a reverse gene mutation assay in bacteria (OECD 471), the test material (CAS 68783-96-0) was applied (doses of 250, 500, 1000, 2500 and 5000 μg/plate in the initial assay, and 1000, 2000, 3000, 4000 and 5000 in the repeat assay) in agar to the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Sanitised, L., 1995). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the test material, and DMSO was the vehicle for the positive controls (9-aminoacridine, 2-nitrofluorene and 2-aminoanthracene and N-methyl-N-Nitro-N-Nitrosoguanidine). Prior to study initiation the solubility of the test substance in the vehicle (tetrahydrofuran, 2 vehicle controls for each strain) was confirmed. In addition, one non-treated control and a positive control were tested for each strain. In the main study there were two treatment sets for each tester strain, with and without metabolic activation.

Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were verified by repeating the assay at dose levels of 1000, 2000, 3000, 4000 and 5000 μg/plate. After 2 days of incubation all plates in the initial and repeat assays were evaluated for gross-toxic effects and total revertant colony numbers. The test substance did not induce significant increases in revertant colonies (equal to or greater than three times the THF control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays.

In a key mammalian cell gene mutation assay (OECD 476), mouse lymphoma L5178Y cells were exposed to a calcium sulfonate read across substance (CAS 68783-96-0), at concentrations of 0, 500, 1000, 1500, 2000, 4000 and 5000 μg/mL with and without S9 metabolic activation (Sanitised, D., 1984). As vehicle DMSO was used, as positive controls 7, 12-dimethylbenzanthracene and ethylmethanesulfonate were used. Prior to study initiation the solubility of the test substance and of the positive control materials in the vehicle (DMSO) was confirmed. A pre-test dose range finding study was conducted at concentrations up to 10,000 μg/mL with and without metabolic activation. In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. DMBA (positive control) was tested with activation and EMS (positive control) was tested without activation. The test material was added to cells with and without activation and incubated for four hours. Cells were then washed and placed in suspension cultures for two days with a cell population adjustment at 24 hours. The cells were then plated in a restrictive media containing trifluorothymidine (TFT) which allows TK-/-cells to grow. Cells were also plated in a non-restrictive media that indicated cell viability. Plates were incubated at 37°C in a humidified 5 % CO2 atmosphere for 10-12 days. Following incubation all plates were scored for total number of colonies/plate. The frequency of mutation by dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. None of the cultures treated with test material with or without activation exhibited mutant frequencies significantly different from the average mutant frequency of the negative (solvent) controls at a per cent total growth of 10% or greater. Positive and vehicle control group responses were appropriate and met the criteria outlined above. So the test substance was not mutagenic in this assay with or without metabolic activation.

In a key mammalian cell cytogenetics assay [chromosome aberration] (OECD 473), Chinese hamster ovary (CHO, clone tested WBL) cell cultures were exposed to the read across substance calcium sulfonate substance, (CAS 68783-96-0), at concentrations of 0, 10, 20, 40, 80, 120 and 160 μg/mL with and without S9 metabolic activation according to OECD 473. As vehicle Tetrahydrofuran (THF, for the test material) and acetone (for the positive controls) was used. Positive controls induced the appropriate response.  There was no evidence of chromosome aberrations induced over background with the test substance. In the initial 16-hours harvest there were statistically significant increases with dose in the % of aberrant cells for both activated and non-activated tests. These trends were not reproducible in the repeat 16-hours harvest and were therefore not considered biologically significant.


Justification for selection of genetic toxicity endpoint
In a key mammalian cell cytogenetics assay [chromosome aberration], Chinese hamster ovary (CHO, clone tested WBL) cell cultures were exposed to the read across substance calcium sulfonate substance, (CAS 68783-96-0), at concentrations of 0, 10, 20, 40, 80, 120 and 160 μg/mL with and without S9 metabolic activation according to OECD 473. As vehicle Tetrahydrofuran (THF, for the test material) and acetone (for the positive controls) was used. Positive controls induced the appropriate response.  There was no evidence of chromosome aberrations induced over background with the test substance. In the initial 16h harvest there were statistically significant increases with dose in the % of aberrant cells for both activated and non activated tests. These trends were not reproducible in the repeat 16h harvest and were therefore not considered biologically significant.

Justification for classification or non-classification

Read across substances were negative for mutagenicity in two in vitro bacterial reverse mutation assay conducted according to OECD 471, an in vitro chromosome aberration assay conducted according to OECD 473, and an in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay conducted according to OECD 476. Therefore, based on the weight of evidence from the available data, benzene, mono-C10-13-alkyl derivatives, distillation residues, sulfonated, sodium salt does not warrant classification for mutagenicity.