Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study has been conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1. However as this study is used in a read-across approach Klimisch 2 is assigned.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

aroclor

Test concentrations with justification for top dose:

0.1, 0.33, 1.0, 3.33 and 10 mg/plate

Vehicle / solvent:

pluronic F127 25 % w/w in ethanol

Negative solvent / vehicle controls:

yes

Remarks:

Pluronic F127 25% w/w in ethanol

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene and ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3/dose group/strain/treatment set

Evaluation criteria:

Number of revertant colonies.

Statistics:

Mean revertant colony count and standard deviation were determined for each dose point.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No reduction in the number of revertants/plate was observed in the range-finding study with strains TA100 and WP2uvrA.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the dose range-finding study with strains TA100 and WP2uvrA with or without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No cytotoxic response was seen in the dose range finding study.

The positive control exhibited at least a 3 fold increase in revertant colonies.

Results: The test substance was not genotoxic in this assay with or without metabolic activation.

Remarks

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, (10,000 µg/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. The calcium sulfonate 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 calcium sulfonate 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.

Conclusions:

Interpretation of results: negative
The calcium sulfonate test material was not mutagenic to Salmonella and E. Coli strains with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to an analogue of C20-C24 alkaryl calcium salt derivative (Analogue of CAS 70024 -69 -0), at concentrations of 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (US-EPA, 1989, Ames Test according to OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of the calcium sulfonate 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, (10,000 µg/plate) and concentrations of S9 mix of 25400 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 the calcium sulfonate 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 37oC. The condition of the bacterial background lawn was evaluated for cytotoxicity and calcium sulfonate test article precipitate. The calcium sulfonate test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation the calcium sulfonate test material was visible at all dose levels in the top layer. The calcium sulfonate 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 calcium sulfonate test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Reference 2

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:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and to GLP, so therefore meets the criteria of Klimisch code 1. However, as this study is used in a read-across approach Klimisch 2 is assigned.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y-3.7.2C mouse lymphoma cells
Cells were stored frozen in liquid nitrogen. Cultures were incubated at 37°C with shaking. Cultures were diluted daily to a cell density of approximately 3 x 10E5 cells/mL. Cultures were checked for bacterial and fungal contamination. Prior to use cultures were treated with methotrexate to reduce the frequency of spontaneously occurring TK-/cells.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 cells

Test concentrations with justification for top dose:

500, 1000, 1500, 2000, 4000, and 5000 µg/mL

Vehicle / solvent:

DMSO - 10 µL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

5 µg/mL (with activation)

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

744 µg/mL (without activation)

Details on test system and experimental conditions:

Exposure method: dilution
The calcium sulfonate test material was suspended in culture and placed on a restricted media containing trifluorothymidine.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
DURATION
- Preincubation period: 4 hours
- Exposure duration: 10-12 days
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY: Method: relative total growth

Evaluation criteria:

Number of colonies/plate.

Statistics:

Mean and standard deviation.

Species / strain:

mouse lymphoma L5178Y cells

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:

RANGE-FINDING/SCREENING STUDIES: Significant toxicity (<90% total growth) at 500 µg/mL occurred both with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The calcium sulfonate test substance was not mutagenic in this assay with or without metabolic activation.

The dose rangefinding study indicated significant toxicity (<90% total growth) at 500 µg/mL with and without metabolic activation. Based on these results the calcium sulfonate test material was evaluated for mutagenicity at concentrations ranging from 500 to 5000 µg/mL. Six cultures with and without activation were selected for cloning at 500, 1000, 1500, 2000, 4000 and 5000 µg/mL. 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 percent total growth of 10% or greater. Positive and vehicle control group responses were appropriate and met the criteria outlined above.

Conclusions:

Interpretation of results: negative
The calcium sulfonate test material was not genotoxic under the conditions of test.

Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to a petroleum derived calcium salt (CAS 68783 -96 -0), at concentrations of  500, 1000, 1500, 2000, 4000 and 5000 µg/mL in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

Reference 3

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:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study meets the criteria for Klimisch code 1. However, as this study is used in a read-across approach, Klimisch 2 is assigned.

Qualifier:

according to guideline

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)

Details on mammalian cell type (if applicable):

Clone Tested: WBL
Cells were thawed and cultured in McCoy's 5A Medium containing 10% fetal bovine serum and 2 mM L-glutamine at 37oC, in 4-6% CO2 in air. Cultures were seeded at 1.2 x 10E6 cells (16-hour harvest) and 0.8 x 10E6 (40-hour harvest) approximately 1 day prior to dosing. Fetal bovine serum was excluded from activated cultures.

Metabolic activation:

with and without

Metabolic activation system:

S9 aroclor incuded rat liver

Test concentrations with justification for top dose:

10, 20, 40, 80, 120, 160 µg/mL (a 50 µL sample was evaluated with and without metabolic activation).

Vehicle / solvent:

tetrahydrofuran for the calcium sulfonate test material (5 µg/mL with and without activation)
acetone for positive control (5 µg/mL with and without activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, 5 µg/mL with and without activation; Tetrahydrofuran, 5 µg/mL with and without activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG)

Remarks:

N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL without activation; 7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL with activation

Details on test system and experimental conditions:

Exposure Method: Dilution
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

Evaluation criteria:

cells with aberrant chromosomes.

Statistics:

The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes/cell was not analyzed. Positive control groups were compared to vehicle control by Fisher Exact Test. Each pair of replicates was compared by Fisher Exact Test. Differences between control and treated groups were compared using Fisher Exact Test and if necessary a 2x2 Fisher Tests. A permutation test was performed to test for dose related trends. Significance levels of less than 0.05 were reported.

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 cloudiness 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'.

Results

The calcium sulfonate test substance was not mutagenic in this assay with or without metabolic activation.

Remarks

In the culture medium solubility test precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/mL and greater. In the pretest toxicity assay there was an 81% reduction (compared to vehicle control) in cell survival at 160 µg/mL without metabolic activation. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 µg/mL. A greater than 50% reduction in cell survival and/or mitotic index was not observed in either the initial or repeat assays. Precipitation was observed at concentrations greater than 40 µg/mL in the chromosomal aberration assay. Therefore, 40 µg/mL was considered to be the limit of solubility for the calcium sulfonate test substance and was selected as the highest test concentration to be evaluated. There were no statistically significant differences in the number of chromosomal aberrations between the treated and vehicle control groups in either the initial or repeat assay at any dose level evaluated (10, 20 and 40 µg/mL with and without metabolic activation). In the initial 16-hour harvest, there were statistically significant increases with dose in the percent of aberrant cells for both the activated and nonactivated evaluations. These trends were not reproducible in the repeat 16-hour harvest and therefore were not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control groups have frequencies of aberrations outside the normal range of the vehicle control and at least twice the vehicle control value.

Conclusions:

Interpretation of results: negative
The calcium sulfonate test material was not clastogenic or 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 (CAS 68783 -96 -0) 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no genetic toxicity studies for Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts (generic name:C10 -13 barium sulfonate) available. Therefore, data on structurally similar analogues is taken to cover information requirements for this endpoint.

Genetic toxicity data (in vitro) for read across substances:

In the key study, a reverse gene mutation assay in bacteria, 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 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (Sanitised, H., 1989, OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of calcium sulfonate 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 calcium sulfonate test article, (10,000 µg/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 calcium sulfonate 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 the calcium sulfonate 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 calcium sulfonate test article precipitate. The calcium sulfonate test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation calcium sulfonate test material was visible at all dose levels in the top layer. The calcium sulfonate 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 calcium sulfonate test substance (Analogue of CAS 70024 -69 -0) 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 with a calcium sulfonate read across substance, (CAS 68783-96-0), the calcium sulfonate test material 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, according to OECD 471). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the calcium sulfonate 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 calcium sulfonate 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 calcium sulfonate test substance (CAs 68783 -96 -0) 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 supporting study with a calcium sulfonate read across substance, (CAS 61789-86-4), the calcium sulfonate test material was applied (All strains - except TA1537 - with and without S9 (-S9) at 5000 1500, 500, 150, 50 and 15 µg/plate, and at 2500, 750, 250, 75, 25 and 7.5 µg/plate for TA1537 only without S9) in agar to the S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 (Loveday, K.S., 1988, according to OECD 471). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the test material, and DMSO or sterile distilled water was the vehicle for the positive controls (9-aminoacridine, 2-aminoanthracene, benzo(a)pyrene, 4 -nitroquinoline-N-oxide, 4 Nitrofluorene and N-ethyl-N-nitro-N-nitrosoguanidine). Negative control and positive controls were tested for each bacterial strain. The calcium sulfonate test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level and was tested up to the maximum recommended dose level of 5000 µg/plate. No calcium sulfonate test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. The calcium sulfonate test sample (CAS 61789 -86 -4) was considered to be non-mutagenic under the conditions of this test.

 

Mammalian cell gene mutation data (in vitro) for read across substances

In a key mammalian cell gene mutation assay, 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, according to OECD 476). As vehicle DMSO was used, as positive controls 7,12 -dimethylbenzanthracene and ethylmethanesulphonate were used.

Prior to study initiation the solubility of the calcium sulfonate 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 calcium sulfonate 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 calcium sulfonate 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 calcium sulfonate test substance was not mutagenic in this assay with or without metabolic activation.

Chromosome aberration data (in vitro) for read across substances

In a key mammalian cell cytogenetics assay [chromosome aberration], Chinese hamster ovary (CHO, clone tested WBL) cell cultures were exposed to a calcium sulfonate read across substance, (CAS 68783-96-0), at concentrations of 0, 10, 20, 40, 80, 120 and 160 µg/mL with and without S9 metabolic activation (Sanitised, M., 1995, according to OECD 473). As vehicle Tetrahydrofuran (THF, for the calcium sulfonate test material) and acetone (for the positive controls) was used. The solubility of the test substance in the culture medium was established at concentrations of 10, 20, 39, 78, 156, 313, 625, 1250, and 2500 µg/mL. Visual and microscopic examinations were made for precipitation at 0, 30 and 180 minutes post preparation. Concentrations showing signs of insolubility at any of these time points were considered unsuitable for dosing. A Dose range finding study was conducted with the calcium sulfonate test substance and vehicle controls tested in duplicate cultures each with and without activation. Calcium sulfonate test substance tested at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 µg/mL. Cytotoxicity and mitotic indices were evaluated. In the main study there were two treatment sets for each concentration of calcium sulfonate test substance, with (+S9) and without (-S9) metabolic activation. 7,12-Dimethylbenz(a) anthracene - DMBA (positive control) was tested with activation and N-Methyl-N-Nitro-N-Nitrosoguanidine - MNNG (positive control) was tested without activation. Prepared cultures were treated with calcium sulfonate test substance or control material and were incubated for 16 hours. A repeat assay was performed using 16 and 40 hour harvest time points. Vehicle, MNNG and DMBA cultures were incubated for 16 hours only. Two to three hours prior to the 16 and 40-hour harvest the spindle inhibitor, Colcemid, was added to each culture to obtain a final concentration of 0.2 µg/mL. Harvested cells were evaluated microscopically for per cent confluency, morphology and estimated number of mitotic cells prior to harvest. The initial assay and the repeat assay (each after 16-hr incubations) with and without S9 were conducted with 10, 20 and 40 µL/mL. The repeat assay with and without S9 (after 16 and 40-hr incubations) was conducted with 10, 20 and 40 µg/mL. Slides were prepared for these groups using Giemsa stain. Two slides/treatment groups were evaluated. 200 metaphase cells (100 per culture) each containing 19-23 chromosomes per treatment group were scored. Chromosomes were counted for each cell. Chromosome aberrations, either chromosome or chromatid type were recorded. The following observations were recorded and excluded from the total aberration frequency: gaps, polyploid and endoreduplicated cells, pulverized chromosomes, Robertsonian translocations, translocations and abnormal monocentric chromosomes. The percentage of aberrant cells and the frequency of aberration (%) per treatment group were determined. In order for a test substance to be considered to have induced a positive response compared to vehicle control a statistically significant dose related increase in the percentage of aberrant cells along with a mean percentage of aberrant cells in excess of 5% in at least one treatment group were required. Or, a reproducible and statistically significant response in at least one treatment group with a mean % of aberrant cells exceeding 5% was observed. Calcium sulfonate test substance concentration verification was performed on the highest stock concentration in both the initial and repeated assays. Results were within 6% of nominal. Samples were homogeneous and stable for the intended period of use. In the culture medium solubility test precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/mL and greater. In the pre-test toxicity assay there was an 81% reduction (compared to vehicle control) in cell survival at 160 µg/mL without metabolic activation. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 µg/mL. A greater than 50% reduction in cell survival and/or mitotic index was not observed in either the initial or repeat assays. Precipitation was observed at concentrations greater than 40 µg/mL in the chromosomal aberration assay. Therefore, 40 µg/mL was considered to be the limit of solubility for the calcium sulfonate test substance and was selected as the highest test concentration to be evaluated. There were no statistically significant differences in the number of chromosomal aberrations between the treated and vehicle control groups in either the initial or repeat assay at any dose level evaluated (10, 20 and 40 µg/mL with and without metabolic activation). In the initial 16-hour harvest, there were statistically significant increases with dose in the percent of aberrant cells for both the activated and non-activated evaluations. These trends were not reproducible in the repeat 16-hour harvest and therefore were not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control groups have frequencies of aberrations outside the normal range of the vehicle control and at least twice the vehicle control value. The calcium sulfonate test material (CAS 68783 -96 -0) was not clastogenic or genotoxic under the conditions of this study.

Data on DNA damage and/or repair - micronucleus assay (in vivo) for read across substances

In the key in vivo study, a mouse bone marrow micronucleus assay, Swiss Albino Crl:CD-I (ICR) BR mice were treated once via the peritoneum with a calcium sulfonate read across substance (Analogue of CAS 70024-69-0), at doses of 0, 100, 200, 400 and 500 mg/kg bw (Sanitised, I., 1989, OECD 474). Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.  A range-finding study was conducted at 200, 400 and 600 mg/kg. Mortality and physical observations were evaluated. During the dose range-finding study mortality (9 of 10 animals) was observed at 600 mg/kg but not at lower dose levels. Signs of toxicity observed at all dose levels included reduced faeces, reduced food consumption, hyperactivity and phonation. Decreased motor activity was observed at 400 and 600 mg/kg. Based on these results dose levels of 100, 200, 400 and 500 mg/kg were selected for the main study. In the main study the vehicle was peanut oil and the dose volume 5 mL/kg. As positive control substance triethylenemelamine was used. All animals were observed frequently for physiological or behavioural abnormalities on the day of dosing and at least twice daily thereafter. Body weights were taken on the first day of the study prior to treatment and at sacrifice. Macroscopic pathology performed on all animals at sacrifice. Five/sex from each treatment group and vehicle control group were sacrificed for bone marrow sampling 24, 48 and 72 hours post treatment. Positive controls sampled at 24 hours only. NCE/PCE ratio and %PCE of total erythrocytes were calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. The number of micronuclei in NCEs was also determined. During the main study, toxicity was observed at 400 and 500 mg/kg. At 500 mg/kg 5 males and 4 females of 15/sex died prior to the scheduled sampling time. At 400 mg/kg 1 of 18 treated females died on day 3. Other clinical signs of toxicity included palpebral closure, decreased motor activity and weakness. Cytotoxicity was observed in both sexes. A statistically significant increase in NCE/PCE ratio was observed in males at 500 mg/kg at 24 hours. Elevated ratios were also observed in individual animals of both sexes in other groups. Altered proportions of erythrocytes to nucleated cells were noted for both sexes in the treated groups. No biological or statistical significant increase in the number of micronucleated-PCE was observed in any treated group compared to the vehicle control. All values for individual animals were within the expected range of micronucleated-PCE/1000 PCE expected for control animals. The variability in response observed in the treated animals was similar to that observed in the vehicle controls. The positive control exhibited a statistically significant increase in micronuclei as expected. Chemical analysis confirmed that the dosing solution preparation procedure utilized for this study resulted in homogeneous solutions of appropriate concentration. As there was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time, under the conditions of this study the calcium sulfonate test material (Analogue of CAS 70024 -69 -0) did not induce micronuclei in bone marrow erythrocytes of mice.

In a supporting study, a mouse bone marrow micronucleus assay, Swiss Albino mice were treated trice (three treatments approximately 24 h apart followed by sacrifice 24 h after final dose) via oral gavage with a calcium sulfonate read across substance (CAS 68783-96-0), at doses of 0, 500, 1000 and 2000 mg/kg bw in peanut oil (Sanitised, N., 1995, OECD 474). Bone marrow cells were harvested at 24 hours after the last treatment. A range-finding study was conducted at 500, 1000 and 2000 mg/kg. Mortality and physical observations were evaluated. All dose range-finding animals survived and were free of clinical signs. Bone marrow toxicity was not observed at any dose levels tested. Therefore 2000 mg/kg was selected as the high dose for the micronucleus assay. The mid and low doses were selected to be 1/2 and 1/4 of the high dose. In the main study the vehicle was peanut oil and the dose volume 5 mL/kg. As positive control substance cyclophosphamide was used. All animals were observed frequently for physiological or behavioural abnormalities on the days of dosing and at least twice daily thereafter. Five/sex from each treatment group and vehicle control group were sacrificed for bone marrow sampling 24 h after the last treatment. NCE/PCE ratio and %PCE of total erythrocytes were calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. The number of micronuclei in NCEs was also determined. In the main study, all vehicle, positive control and treated animals were normal after dosing and remained healthy until sacrifice. There were no dose related increases or statistical differences in micronuclei formation observed at any dose level. Cytotoxicity was not observed since there was no statistically significant decreases in the percentage of polychromatic erythrocytes compared with the vehicle control. The positive control induced a statistically significant increase in mean micronucleated PCEs in both sexes compared with the vehicle controls which indicated the positive control was clastogenic and responded appropriately. The positive control also induced cytotoxicity. Chemical analysis confirmed the uniformity and stability of the calcium sulfonate test material in peanut oil for at least 9 days at all 3 concentrations. Concentration verification analysis confirmed that each dose level was within 3% of nominal concentration. The calcium sulfonate test substance (CAS 68783 -96 -0) was not genotoxic under the conditions of the study.

In another supporting study, a mouse bone marrow micronucleus assay, B6C3F1 mice were treated once via oral gavage with a calcium sulfonate read across substance (Analogue of CAS 61789-86-4), at the doses of 5 g/kg undiluted (Loveday, K.S., 1988). A total of 80 mice, 40 males and 40 females, were used for the study. Ten animals (5 males and 5 females) were used per test group. Bone marrow cells were harvested at 18, 24 and 48 hours after the last treatment.  In the main study no vehicle was used and the dose volume did not exceed 10 mL/kg. Extra heavy U. S. P. mineral oil was used as the negative control. As positive control substance cyclophosphamide was used. Cyclophosphamide is tested at 50 and 75 mg/kg in female mice and at 25 and 50 mg/kg in male mice to ensure a positive response in both sexes. Only one positive control group is analysed for each sex. One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in this animal. Following each exposure period (18, 24 and 48 hours), mice were killed by cervical dislocation and the femurs were removed and the ends cut. Bone marrow cells were collected by flushing the cavity with a phosphate buffered saline solution (pH 7.4). The cells are concentrated by centrifugation and the cell pellet resuspended in a small volume of phosphate buffer. The cells are smeared on clean slides and air-dried. The slides are stained with acridine orange (0.125 mg/mL). Slides were made by placing one drop of suspension on a clean slide and spreading the cells evenly with a second slide. At least two slides were made from each animal. Slides were stained with acridine orange a few minutes prior to analysis using a fluorescent microscope. Slides are scored using a fluorescent microscope (FITC filter combination, excitation wavelength 495 nm, emission wavelength 520 nm). One thousand polychromatic erythrocytes (PCE) from each animal are scored for micro-nuclei. The ratio of PCE to normochromatic erythrocytes (NCE) are determined for each animal by counting a minimum of 1,000 erythrocytes. The NCE are not scored for micronuclei. The mean micronucleated PGEs for the negative control male mice were 2.6, 3.0 and 2.4 per 1000 PGEs for the three time periods. The average number of micronucleated PCEs for the positive control male mice was 14.5. The mean micronucleated PCEs for the calcium sulfonate test chemical were 1.7, 4.3 and 3.6 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values. The mean number of micronucleated PCEs for the negative control female mice were 1.9, 2.1 and 2.9 per 1000 PCEs for the three time periods, while the positive control female mice had an average of 20.5. The frequencies of micronucleated PCEs in female mice treated with the calcium sulfonate test chemical were 2.9, 3.2 and 2.1 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values. Based on these data, the calcium sulfonate read across substance, which was tested at the maximum dose of 5 g/kg, did not induce a significant increase in micronuclei in bone marrow cells from either male or female B6G3Fl mice. Thus, the calcium sulfonate read across substance (61789-86-4) was negative in this in vivo assay.

Justification for selection of genetic toxicity endpoint
No study is selected since all results are negative

Short description of key information:
Genetic toxicity in vitro, OECD 471, CAS 70024-69-0, negative
Mammalian cell gene mutation, OECD 476, CAS 68783-96-0, negative
Chromosome aberration, OECD 473, CAS 68783-96-0, negative
Micronucleus test, OECD 474, CAS 70024-69-0, negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the all in vitro and in vivo data available for the calcium sulfonate read across substances, it is clear that all calcium sulfonate read across substances are neither mutagenic, nor genotoxic, nor induced micronuclei. Therefore also the barium sulfonate target substance (mono-C10 -C13) is considered to be not mutagenic nor genotoxic. According to the Regulation (EC) No 1272/2008 (CLP Regulation), Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts, due to the high structural similarity of these substances does not meet the criteria for classification and will not require labelling as a mutagen.